Rapid Diagnostic Test for LAMP

ABSTRACT

Kits and methods are described that are directed to specific and sensitive methods of target nucleic acid detection and more specifically detecting target nucleic acids directly from biological samples. The kits and methods were developed to be easy to use involving a minimum number of steps and giving rapid and consistent results either at point of care or in high throughput situations. The kits and methods utilize in various combinations, reversible inhibitors of kit components, thermolabile enzymes, poloxamers, various salts, indicators and one or more Loop-Mediated Isothermal Amplification (LAMP) primer sets for detecting single and/or multiple targets and variants of the targets including SARS-CoV-2 targets and variants thereof in a single reaction. The kits and methods permit detection of the target nucleic with similar sensitivity regardless of the presence of undefined mutations that may enhance the virulence of cells or viruses containing the undefined mutations.

CROSS REFERENCE

This application is a continuation in part of U.S. application Ser. No.17/221,451 filed Apr. 2, 2021, which is a continuation of U.S.application Ser. No. 17/122,979, filed Dec. 15, 2020, which claims rightof priority to U.S. Provisional Application No. 62/988,696, filed Mar.12, 2020; U.S. Provisional Application No. 63/001,909, filed Mar. 30,2020; U.S. Provisional Application No. 63/013,442, filed Apr. 21, 2020;U.S. Provisional Application No. 63/022,303, filed May 8, 2020; U.S.Provisional Application No. 63/027,216, filed May 19, 2020; U.S.Provisional Application No. 63/048,556, filed Jul. 6, 2020; and U.S.application Ser. No. 16/938,575, filed Jul. 24, 2020.

This application also claims right of priority to U.S. ProvisionalApplication No. 63/068,564, filed Aug. 21, 2020; U.S. ProvisionalApplication No. 63/106,120, filed Oct. 27, 2020; and U.S. ProvisionalApplication No. 63/165,465, filed Mar. 24, 2021. The contents of theseapplications are incorporated herein by reference in their entirety.

BACKGROUND

The ability to detect an infectious agent in a widespread epidemic is acritical aspect of successful quarantine efforts and enables screeningof potential cases of infection from patients in a clinical setting.Enabling testing outside of sophisticated laboratories broadens thescope of control and surveillance efforts, but also requires robust andsimple methods that can be used without expensive instrumentation.

The emergence of a new coronavirus (2019-nCoV, also called SARS-CoV-2and COVID-19) has caused a world-wide pandemic for which diagnostictests play a critical role. The current diagnostic standard combinesclinical symptoms and molecular method, where symptoms for some patientsrange from life threatening to resembling those of common cold andinfluenza, to no symptoms or widely variable symptoms. Monitoring thespread of infection requires accurate, easy to use, widely available andcost sensitive molecular diagnostic tests. These molecular methodsinclude metagenomics sequencing mNGS and reversetranscriptase-quantitative PCR (RT-qPCR) (Huang, et al. (2020) Lancet,395, 497-506). mNGS is restricted by throughput, turnover time, highcosts and requirement for high technical expertise. RT-qPCR requiresmultiple steps and expensive laboratory instruments and is difficult toutilize outside of well-equipped facilities. A rapid, specific, andsensitive diagnostic single test for one or several pathogens would bedesirable to identify and track infected humans, animals or plants in awidespread epidemic that might threaten health and well-being.

Target pathogens, and particularly RNA viruses, naturally displaymutations and changes in their genomic sequences that can impact thesensitivity and accuracy of the molecular diagnostic test when themutations occur in the regions targeted by the oligonucleotide primersand/or probes. The ongoing SARS-CoV-2 pandemic has seen the emergence ofnumerous viral variants from different regions of the world, withprominent effects on detection using molecular assays. For example, theB.1.1.7 “alpha” variant features a 6-base deletion (removing 2 aminoacids of the spike protein, Δ69-70) which causes a failure of the S Geneassay in the widely used TaqPath™ COVID-19 multiplex RT-qPCR test(Vogels et al. PLoS Biol 19(5), e3001236 (2021)). The other targets inthis assay are unaffected, resulting in the ability to provide potentialidentification of variant RNA during detection, though sequencing isnecessary for proper variant identification. In this particular case theother targets can be used for diagnostic detection, but the sensitivityof RT-qPCR to variant mutations is a significant concern to diagnostictesting given the worldwide reliance on the method.

While specific mutations like the alpha Δ69-70 are characterized ingreat detail, systematic evaluation of potential mutation effects ondiagnostic assay performance is lacking. Mutations could be single basechanges or deletions that remove a small region of the genome and couldresult in changes of the amino sequences (missense mutation) or nochange (silent mutation). Larger deletions like Δ69-70 likely have moredetrimental effects than single base changes, though single base changeslocated close to the 3′ ends of primers might impact more than thoselocated near the 5′ ends. The widely-used TaqMan® probes rely onspecific hybridization to targeted sequences and may display greatersensitivity to mutations than when they occur in primer binding regions;the Δ69-70 deletion occurs in the TaqPath probe region and completelyprevents detection of RNA with that deletion.

SUMMARY

In one aspect, there is provided a master mix comprising: a DNApolymerase suitable for isothermal amplification of DNA; dNTPs (dATP,dGTP, dCTP, and dTTP); and at least one dye that changes color orfluorescence in response to DNA amplification. The master mix may bedried or may be in a weakly buffered solution. In many embodiments, themaster mix is a LAMP master mix. However, the parameters tested hereinmay be applicable to master mixes for non-LAMP isothermal amplificationreactions.

The DNA polymerase in the master mix may be any polymerase suitable foruse in an isothermal amplification reaction (e.g., in a Loop-MediatedIsothermal Amplification (LAMP) reaction). Suitable DNA polymerases areknown in the art and include strand displacing DNA polymerasespreferably mesophilic DNA polymerases such as Bst polymerase or variantsthereof or Bsu DNA polymerase or variants thereof. A reversibleinhibitor of the DNA polymerase may be included in the master mix. Forexample, the reversible inhibitor may inhibit the polymerase attemperatures below 50° C. Examples of reversible inhibitors includeaptamers and antibodies.

The at least one dye may be a colored dye detectable in visible light,or may be a fluorescent dye, so long as the dye provides a change insignal (e.g., a change in color, or a change in color intensity orfluorescence intensity) in response to an amplification reaction.

In one embodiment, the at least one colorimetric or fluorescent dye isor includes a dye that is pH sensitive. When the master mix containingthe dye is used in an amplification reaction that alters the pH of thereaction mix, the spectral or fluorescent properties of the dye change(e.g. the dye changes color), which provides confirmation thatamplification has occurred. Examples of pH sensitive dyes includecolorimetric dyes such as phenol red, cresol red, m-cresol purple,bromocresol purple, neutral red, phenolphthalein, naphtholphthein, andthymol blue; and fluorescent dyes such as2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein or a carboxylseminaphthorhodafluor (e.g. SNARF-1). In one embodiment, the at leastone pH sensitive dye is phenol red.

In one embodiment, the at least one dye is or includes a dye that is notpH sensitive, such as a metallochromic indicator. When the master mxcontaining the metallochromic indicator dye is used in an amplificationreaction that alters the availability of one or more metal ions in thereaction mix, the spectral or fluorescent properties of the dye change(e.g. the dye changes color), which provides confirmation thatamplification has occurred. In one embodiment, the metallochromicindicator dye is 4-(2-pyridylazo) resorcinol (PAR). If PAR is used, themaster mix may additionally comprise manganese ions such that the PAR inthe master mix is complexed with Mn ions to form a PAR-Mn complex.Another example of a metallochromic dye is hydroxynaphthol blue(Wastling et al. (2010) PLoS Negl Trop Dis 4(11): e865. doi:10.1371/journal.pntd.0000865).

In a preferred embodiment, the visually detectable dye in the reactionmix (1× master mix combined with sample) is in the range of 50 μM-200μM.

The master mix for LAMP may optionally further comprise at least one setof primers (e.g., two, three, four, or five sets of primers) havingspecificity for a target nucleic acid. If multiple primer sets are used,each primer set may target a different nucleic acid sequence within thetarget nucleic acid (e.g., two or more different viral gene sequences).For example, the master mix may comprise at least two sets of primers,each specific for a different SARS-CoV-2 target sequence (e.g., in theORF 1a gene and/or Gene N of SARS-CoV-2). Frequently it is preferredthat the primers are provided separately from the master mix.

The master mix may comprise a reverse transcriptase, such as an HIVderived reverse transcriptase, an intron encoded reverse transcriptase,or a reverse transcriptase variant of Moloney murine leukemia virus. Themaster mix may comprise a reversible inhibitor for the reversetranscriptase that inhibits activity of the enzyme at or below 40° C.Examples of reversible inhibitors include one or more inhibitoryoligonucleotides or antibodies. One or more inhibitors of RNase activitythat are not reversible may be included in the master mix for inhibitingRNase activity. Examples include inhibitory oligonucleotides such asinhibitory oligonucleotides such as aptamers, or antibodies forinhibiting RNase A and an aptamer for inhibiting RNase I. The master mixmay include an RNAse inhibitor that is not an aptamer. The master mixmay comprise dUTP and/or a uracil DNA glycosylase (UDG), such as athermolabile UDG. In one embodiment, the master mix optionally comprisesa molecule comprising C—(NH₂)₂NH; such as guanidine hydrochloride,guanidine thiocyanate, guanidine chloride, guanidine sulfate, orarginine. In one embodiment, the master mix comprises guanidinehydrochloride. The C—(NH₂)₂NH containing molecule can be present in themaster mix at a concentration in the range of up to 60 mM, such as inthe range of 20 mM-40 mM (e.g., about 20 mM, 30 mM or 40 mM).

However, in certain embodiments, the master mix does not contain amolecule comprising C—(NH₂)₂NH. In these circumstances, a compositioncomprising C—(NH₂)₂NH; such as guanidine hydrochloride, guanidinethiocyanate, guanidine chloride, guanidine sulfate, or arginine is notincluded in the master mix may be provided separately such as containedin a lysis buffer or in a mixture containing primer sets for adding to aLAMP reaction mixture. In one embodiment, a concentration of guanidinesalt in the LAMP reaction mixture is preferably 40 mM guanidine salt.The C—(NH₂)₂NH containing molecule can be present in the composition ata concentration in the range of up to 60 mM, such as in the range of 20mM-40 mM (e.g., about 20 mM, 30 mM or 40 mM). In certain embodiments,the master mix contains primers that are specific for a targetpolynucleotide. In other embodiments, the master mix does not containprimers where these are added separately.

In embodiments where the master mix is dried, the master mix may befreeze dried, air dried, or lyophilized. The master mix may beimmobilized, for example on paper, or on a natural or synthetic polymer.The dried master mix is reconstituted prior to use in an amplificationreaction.

In embodiments where the master mix is in solution (e.g., followingreconstitution), the master mix is in a weakly buffered solution, suchas in a Tris buffer. The weakly buffered solution preferably has aconcentration less than 5 mM, such as less than 5 mM Tris or equivalentbuffer. In one embodiment, the weakly buffered solution is in the rangeof 0.5 mM to 5 mM, such as 0.5 mM to 5 mM Tris or equivalent buffer. ThepH of the master mix may be buffered in the range of pH 7.5-pH 9.0; suchas in the range of pH 7.8-pH 8.5, or pH 8.1-pH 8.5. The liquid form ofthe master mix may be in any suitable reaction container.

In one aspect there is provided a kit comprising a master mix asdescribed herein. The kit may optionally further comprise a heatingblock or water bath suitable for heating a reaction tube, plate, orpaper, or a plurality of the same to a temperature suitable forisothermal amplification.

In one aspect there is provided a method for determining whether atarget nucleic acid is present in a sample, comprising: bringing analiquot of the sample into contact with a master mix as described hereinto form a reaction mixture wherein the reaction mix additionallyincludes primers that are specific for the target nucleic acid. In oneexample, the sample may be in an aqueous solution or absorbed to a testmatrix such as paper. The master mix may include additional reagents toenhance sensitivity of the assay such as a guanidine salt, or this couldbe added separately to the reaction. Additionally, for LAMP, one primerset or a plurality of primer sets may be included in the master mix oradded separately to the reaction mix. Not all target specific primersets work equally well. For improved sensitivity, it is desirable totest a number of primer sets to select one or a combination of primersets to maximize sensitivity of the LAMP assay. Determination as towhether the target nucleic acid is present in the sample may thenproceed by detecting a change in the spectral properties, color, orfluorescence of the reaction mixture.

In one embodiment, the method involves isothermal amplification of thetarget nucleic acid in a LAMP reaction or a helicase-dependentamplification reaction (HDA). In one embodiment, the method involvescolorimetric LAMP, which may be pH sensitive or may be pH insensitive(e.g., using PAR). In one embodiment, the method uses two, three, four,or five sets of target-specific primers in a multiplexed reaction (e.g.,multiplexed LAMP); wherein the primers are added to the reaction mixturein the master mix or are added separately.

The sample may be a clinical sample, such as a sample of a body fluid(e.g., blood, sputum, saliva, mucous, lymph, sweat, urine, feces, etc.)or a sample taken from a swab such as a nasal, oral, or buccal swab,which may be from a human or other mammalian subject. The sample mayalternatively be an environmental sample. In some embodiments, themethod is performed directly on the sample (e.g., crude tissue or celllysate, or whole blood) without a step of purifying target nucleic acidfrom the sample. To facilitate this, the sample may be added to a lysisbuffer such as exemplified herein for saliva that may nonetheless besuitable for any body fluid. The sample may alternatively be a sample ofpurified nucleic acid.

Where an aqueous solution is referred to without reference to a bodyfluid or environmental sample, it may include sterile water or a weakbuffer (e.g., 0.5 mM to 5 mM, such as 0.5 mM to 5 mM Tris or equivalentbuffer) where the aqueous solution optionally contains a nucleaseinhibitor such as an RNase inhibitor or a DNase inhibitors or both.

The target nucleic acid may be any DNA or RNA of interest. For example,the nucleic acid may be associated with a pathogen or a diagnostictarget for pathogenesis. In one embodiment, the target nucleic acid isRNA or DNA of a target pathogen. In one embodiment, the target nucleicacid is from a bacterium. In one embodiment, the target nucleic acid isfrom a multi-cellular parasite, such as a parasitic nematode. In oneembodiment, the pathogen is a virus; for example, an RNA virus, such asa coronavirus. For example, the pathogen may be SARS-CoV-2. In oneembodiment, the target SARS-CoV-2 RNA sequence is the ORF1a gene and/orGene N or portion thereof. Thus, there is provided a method fordetermining whether a SARS-CoV-2 nucleic acid is present in a sample,comprising: bringing an aliquot of the sample in an aqueous solutioninto contact with a master mix as described herein to form a reactionmixture, wherein the master mix or reaction mixture comprises at leastone set of primers specific for a target SARS-CoV-2 nucleic acid; anddetermining whether the target SARS-CoV-2 nucleic acid is present in thesample by detecting a change in the color or fluorescence of themixture.

In one embodiment, the nucleic acid is associated with gene expression,or may be an indicator of a metabolic response to a pharmaceuticalpreparation or allergen. For example, the method may be for determininga gene expression profile in response to an environmental or metabolicevent, or in response to a therapeutic treatment. In one embodiment, thetarget nucleic acid is DNA, and the method is for determining one ormore genetic loci correlated to a phenotype. For example, the geneticloci may be selected from the group consisting of a single nucleotidepolymorphism (SNP) in a genome, an exon, or a gene in a genome. Thesemethods may be useful in diagnosis of a genetic disease or inpersonalized medicine.

In one embodiment, the method uses a pH-sensitive dye. During nucleicacid amplification, hydrogen ions accumulate in the reaction mixture sothat the mixture becomes increasingly acidic with increasingamplification. pH sensitive dyes change their color, color intensity, orfluorescent intensity, in response to the change in pH in the reactionmixture.

In one embodiment, the method uses PAR as the dye, and the master mix orthe reaction mixture further comprises manganese ions (e.g., about 0.4mM Mn ions per 100 μM PAR). The complex of PAR with manganese ions is ina red-colored state. Pyrophosphate produced during the nucleic acidamplification process, as a byproduct of primer nucleic acidpolymerization, sequesters manganese with a higher affinity than doesPAR, thereby removing the manganese from solution and returning PAR to anon-complexed, yellow-colored state. In one embodiment, the reactionmixture further comprises a non-ionic detergent such as Triton X-100(e.g. at about 1%-4%, such as about 1% or 2%), which is shown herein tofurther enhance the color-change observed when using PAR.

The concentration of the visually detectable dye in the reaction mix maybe in the range of 50 μM-250 μM; such as at 50 μM-150 μM, for example atabout 50 μM, 75 μM, 100 μM, or 150 μM.

In one embodiment, the reaction mixture further comprises a moleculecomprising C—(NH₂)₂NH; such as guanidine hydrochloride, guanidinethiocyanate, guanidine chloride, guanidine sulfate, or arginine. In oneembodiment, the molecule is guanidine hydrochloride. The C—(NH₂)₂NHcontaining molecule can be added to the reaction at a concentration ofup to 60 mM, such as in the range of 20 mM-40 mM (e.g., about 20 mM, 30mM, or 40 mM). If the method uses a C—(NH₂)₂NH containing molecule(e.g., guanidine hydrochloride), the sodium chloride concentration inthe reaction mixture is preferably less than 40 mM; for example, thereaction mixture may contain NaCl at a concentration of about 20 mM orabout 10 mM. The reaction mixture may alternatively, or additionally,comprise KCl at a concentration of less than 100 mM (e.g., less than 40mM).

The method may comprise the step of combining one or more RNaseinhibitors and/or thermolabile Proteinase K with the sample, prior tocombining the sample with the master mix to form the reaction mixture.Alternatively, one or more RNase inhibitors and/or thermolabileProteinase K can be added to the reaction mixture together with thesample, in which case the thermolabile Proteinase K should beinactivated prior to adding the master mix. The method may compriseadding a reverse transcriptase to the reaction mixture (either via themaster mix, or separately) if the target nucleic acid is RNA, such asviral RNA. The reaction mixture may further comprise dUTP and UDG (e.g.,thermolabile UDG), which may be added to the reaction mixture from themaster mix. Alternatively, the dUTP may be added to the reaction mixturefrom the master mix, and the UDG may be added separately to the reactionmixture.

In one embodiment, the method comprises analyzing multiple samples. Forexample, the method may use a reaction container that has multiplecompartments each for analyzing a separate sample.

In one embodiment of the method, the master mix is dried and immobilizedonto e.g., paper, and an aliquot (e.g., droplet) of the sample is addedto the paper, followed by a heating step, resulting in amplification oftarget nucleic acid in the sample.

The change in the spectral or fluorescent properties of the dye can bedetected by eye or using a spectrophotometer or fluorimeter or recordedby means of a camera or other color sensitive recording device. In oneembodiment, the method involves comparing the spectral or fluorescentproperties of the dye before and after amplification has occurred. Inone embodiment, the change in spectral properties or fluorescence of themixture can be recorded by a spectrophotometer having dual wavelengthcapabilities, digitized, and stored by a computer.

In one aspect, there is provided a composition, comprising: one or moreprimer sets suitable for amplification, such as for an isothermalamplification reaction such LAMP, the primer sets having specificity fora single target nucleic acid of interest; and a buffer containing amolecule comprising C—(NH₂)₂NH.

In one embodiment, the composition comprises two, three, four, or fiveprimer sets, each having specificity for a single target nucleic acid ofinterest; such as a viral RNA sequence (e.g., SARS-CoV-2 RNA).

In one embodiment, the composition comprises C—(NH₂)₂NH such as selectedfrom guanidine hydrochloride, guanidine thiocyanate, guanidine chloride,guanidine sulfate, or arginine. In one embodiment, the molecule ispresent in the composition at a concentration in the range of up to 60mM, such as in the range of 20 mM-40 mM (e.g., about 20 mM, 30 mM, or 40mM).

The composition may further comprise one or more reagents selected froma DNA polymerase, such as Bst polymerase; a reverse transcriptase; andan RNAse inhibitor. The composition may comprise dNTPs, which mayoptionally include dUTP; and/or may comprise a thermolabile UDG (alsoreferred to herein as uracil deglycosylase). The composition may furthercomprise a reporter molecule for detecting amplification in the presenceof a target nucleic acid; for example, a colorimetric or fluorescent dyeas described herein (e.g., PAR).

In one aspect there is provided a method of isothermal amplification(e.g., LAMP or HDA), comprising: (a) adding any embodiment of the mastermix described herein that contains a suitable polymerase, and dNTPs to asample comprising a target nucleic acid to form a reaction mixture, andin the presence of suitable primers allowing amplification to occur; and(b) detecting whether the target nucleic acid is present in the sample.

The target nucleic acid may be as described above. In one embodiment,the target nucleic acid is a viral nucleic acid such as viral RNA. Forexample, the target nucleic acid may be a SARS-CoV-2 RNA. In oneembodiment, the target SARS-CoV-2 RNA sequence is located within theORF1a gene and/or Gene N.

In one embodiment, the molecule comprising C—(NH₂)₂NH (e.g., guanidinehydrochloride, guanidine thiocyanate, guanidine chloride, guanidinesulfate, or arginine) is present in the reaction mixture at aconcentration of up to 60 mM, such as in the range of 20 mM-40 mM (e.g.about 20 mM, 30 mM or 40 mM). In one embodiment, the reaction mixturemay further comprise NaCl at a concentration of less than 40 mM; such asat a concentration of about 20 mM. The reaction mixture mayalternatively, or additionally, comprise KCl at a concentration of lessthan 100 mM (e.g., less than 40 mM).

In one aspect there is provided a method for detecting amplification ofa target nucleic acid, comprising: providing an amplification reactionmixture containing a target nucleic acid and a master mix or compositionas defined herein; and detecting a change in the spectral or fluorescentproperties of the dye resulting from amplification of the target nucleicacid. The target nucleic acid may be as described above. In oneembodiment, the target nucleic acid is a viral nucleic acid such asviral RNA. For example, the target nucleic acid may be a SARS-CoV-2 RNA.

Embodiments describe the use of immobilized reagent and lyophilizedreagents in receiving vessels for saliva and other body fluids to assistin streamlining workflows and improving sensitivity of assays.

Additional embodiments include the following:

A kit is provided for Loop-Mediated Isothermal Amplification (LAMP),that includes either separately or combined in a mixture: (a) a stranddisplacing polymerase, for example, a Family A DNA polymerase, forexample a mesophilic bacterial strand displacing polymerase, for examplea bacillus family A strand displacing DNA polymerase capable of copyingDNA at a temperature in the range of 50° C.-68° C.; (b) a reversibleinhibitor of the polymerase, for example, an oligonucleotide reversibleinhibitor also referred to as an aptamer that binds and inactivates thepolymerase at temperatures at or below 50° C. but is released from thepolymerase at temperatures above 50° C., permitting specificamplification with reduced background; (c) a thermolabile UDG that isinactivated at a temperature of above 50° C., a temperature that ispreferably lower than the temperature at which the polymerase is activeand isothermal amplification occurs where in one example, this is in therange of 60° C.-70° C., for example 64° C., 65° C., 66° C., 67° C. or68° C.; (d) nucleoside triphosphates comprising dATP, dGTP, dCTP, anddTTP; and dUTP; and (e) at least one indicator reagent such as ametallochromic dye, a pH sensitive colorimetric dye or a fluorescent dyethat changes color or provides fluorescence if amplification occurs.

If the kit is intended for use with a target nucleic acid that is RNA,the kit may comprise in addition to the above components, a reversetranscriptase for example a virus derived reverse transcriptase, or anintron expressed reverse transcriptase and a reversible inhibitor of thereverse transcriptase that permits the reverse transcriptase to beactive above 50° C. but not below 50° C. so as to reduce background andenhance sensitivity of the LAMP amplification. The reversible inhibitorof the reverse transcriptase and the strand displacing polymerase shouldnot adversely affect either the effectiveness of each inhibitor or thedesired activity of either enzyme at the desired temperature. In oneexample of a combination of reversible inhibitory oligonucleotides oraptamer these were obtained individually as WarmStart® (New EnglandBiolabs, Ipswich, Mass.) and RTX (New England Biolabs, Ipswich, Mass.)and combined and tested as shown herein to provide the desiredsensitivity for detecting as few as 50 viral genomes (SARS-CoV-2) in asaliva sample spiked with virus.

The kit may additionally contain a receiving container for a biologicalsample. For example, the receiving container may be a vessel with a lidor a paper, a microfluidic device or a polymer surface to which thereagents and/or sample are attached and suitable for absorbing reagentsor sample in a liquid form. The receiving container may be a vessel forcombining reagents in a liquid form with samples in a liquid form. Thekit may also contain lysis reagents for combining with the biologicalsample prior to performing LAMP with the kit reagents. These lysisreagents are for adding to or contained in the receiving container forlysing a biological sample to release any target nucleic acids thereinfor amplification by LAMP. The lysis reagents may include a reducingagent such as Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and ametal chelator such as EDTA. The lysis mixture may further include asalt of C—(NH₂)₂NH⁺. The lysis mixture may further include a poloxamer.Additionally, the kit may contain at least one set of LAMP primers. Thekit may contain a plurality of primer sets for amplifying a plurality oftarget nucleic acids in the biological sample such as for exampledifferent respiratory RNA viruses. The kit may contain a plurality ofprimer sets for amplifying a single nucleic acid target such as a viralgenome such as Gene N and Gene E in SARS-CoV-2 in the biological sample.The kit may contain a plurality of primer sets for amplifying a singleor multiple target nucleic acids from single or multiple samples.Pooling samples is one form of high throughput analysis of populationshaving a low infection rate with a pathogen.

Any of the amplification reagents or the reagents in the lysis mix maybe freeze dried or lyophilized preferably excluding salts ofC—(NH₂)₂NH⁺. In addition, or alternatively, any of the specified enzymeand/or oligonucleotide reagents may be immobilized or incorporated on amatrix. If any of the reagents are supplied in a buffer for use with pHdependent colorimetric LAMP, then the buffering capacity should be nogreater than equivalent to 5 mM Tris.

The kit provided herein may include instructions for use with abiological sample where the biological sample is selected from a bodyfluid or tissue, an agricultural sample, a food sample, a waste product,and a pathogen for example, the biological sample is a body fluid ortissue selected from the group consisting of mucous, urine, lymph,blood, saliva, feces, sputum, sweat, semen and biopsy, for example asaliva or a nasal swab. The instructions provide how to test for atarget RNA genome in a single viral strain or in multiple viral strainsor from multiple samples from a population of subjects.

The kit may contain a receiving container for a biological sample, thatincludes a vessel with a lid, the lid containing a solution of indicatorreagent for release into the reaction vessel after lysis of thebiological sample or after LAMP. Instructions for use of the kit mayinclude a method for high sample throughput workflow enabled by the kitthat is partially or completely automated and further comprises arecording device for storing and/or reporting positive sample data afterdetection of a change in color or fluorescence of the indicatorresulting from amplification of the target nucleic acid.

In one aspect, a reaction mixture, is provided that includes athermolabile UDG, a strand displacing polymerase, a reversible inhibitorof the polymerase, and a salt of C—(NH₂)₂NH⁺.

In another aspect, a lysis mixture is provided for releasing an RNA froma biological sample for detection by amplification and/or sequencing,comprising a poloxamer, a reducing agent and a metal chelating agent.

The lysis mixture may further include a salt of C—(NH₂)₂NH⁺.

In another aspect, a master mix is provided that includes a thermolabileUDG, a strand displacing polymerase, a reversible inhibitor of thepolymerase, a reverse transcriptase and a reversible inhibitor of thereverse transcriptase.

In another aspect, a method is provided for amplifying any targetnucleic acid in a biological sample by LAMP, that includes: (a)combining the biological sample with a lysis reagent to form a lysismix; (b) incubating the lysis mix at a temperature of at least 60° C.for a period of time in the range of 3 minutes to 45 minutes; (c)combining an aliquot of the heat treated mix after step (b) withamplification reagents that include a strand displacing polymerase, areversible inhibitor of the polymerase, a thermolabile UDG, nucleosidetriphosphates, and at least one set of LAMP primers that hybridize tothe target nucleic acid, to produce a reaction mix; and (d) incubatingthe reaction mix under amplification conditions for LAMP to permitinactivation of the thermolabile UDG and amplification of the targetnucleic acid. The amplification reagents may further include a reversetranscriptase and a reversible inhibitor of the reverse transcriptase.

The lysis reagent in (a) may include at least one of a salt ofC—(NH₂)₂NH⁺ and a poloxamer to produce a lysis mix. The lysis reagentmay also include a reducing agent and a metal chelating reagent. Whenthe biological sample is combined with the lysis reagent to form a lysismix, the lysis mix may be incubated at 95° C. for 5 minutes to breakopen the sample and release target nucleic acid.

In the method, any of the reagents in the lysis mix may be immobilizedand/or lyophilized or freeze dried. Prior to the addition of thebiological sample. The method may comprise a further step of determiningif amplification has occurred by measuring fluorescence and/or colorchanges of one or more indicators in the amplification reagent mix.

In examples of the method, the biological sample may be saliva, thetarget nucleic acid may be an RNA virus such as a coronavirus and theamplification reagents may include a reverse transcriptase and a reversetranscriptase reversible inhibitor single or multiple sets of LAMPprimers for amplifying at least two sequences in a target DNA or a cDNAof an RNA target such as Gene E and Gene N sequences in SARS-CoV-2,and/or for amplifying multiple target nucleic acids from differentviruses and/or for amplifying target nucleic acids from differentbiological samples or from different animal subjects.

In one embodiment, a method is provided for analyzing a biologicalsample to determine the presence of a target nucleic acid, thatincludes: (a) combining the biological sample with a lysis reagentcomprising a reducing agent, a metal chelator and at least one of aguanidine salt and a poloxamer to produce a lysis mix; and (b)determining the presence of the target nucleic acid by selectivelyamplifying, by means of, for example, LAMP, the target nucleic acid in areaction mix that comprises an aliquot of the lysis mix. The reactionmix in (b) may include an indicator reagent that changes color orprovides fluorescence if amplification occurs, and the method mayinclude the additional step of determining whether the reaction mixcontains the target nucleic acid based on a change in color orfluorescence.

In one embodiment, a method is provided for detecting an RNA virus insaliva or a nasal swab of a subject, that includes (a) collecting salivain a receiving container that comprises: (i) substrate immobilizedoligonucleotides for binding viral RNA and a lysis reagent mixcomprising two or more reagents (for example, 3 or more reagents)selected from a guanidinium salt, a poloxamer, a reducing agent, a DNaseinhibitor, an RNase inhibitor, a detergent, a metal chelator and aproteolytic agent; or (ii) lyophilized substrate immobilizedoligonucleotides for binding viral RNA, and/or one or more lyophilizedreagents contained in a lysis reagent mix, wherein the lysis reagent mixcomprises two or more reagents (for example, 3 or more reagents)selected from a poloxamer, a reducing agent, DNase inhibitor, an RNaseinhibitor, a detergent, a metal chelator and a proteolytic agent,wherein the lyophilized reagents become rehydrated when contacted by thecollected saliva; (b) incubating the receiving container after step (a)at an effective temperature and time to release nucleic acid from anycoronaviruses in the saliva for binding to the immobilizedoligonucleotides; (c) removing the lysis reagent mix from the receivingvessel leaving the coronavirus genome bound to the immobilizedoligonucleotides on the substrate; (d) adding amplification reagents tothe substrate after step (c), wherein amplification reagents comprisereverse transcription reagents and DNA amplification reagents, to make areaction mix; and (e) incubating the reaction mix under amplificationconditions to amplify a cDNA copy of at least a portion of thecoronavirus genome. In this embodiment, the receiving container may beselected from a paper substrate, a microfluidic device or a polymersurface. The reaction mix of (d) in this method may further include anindicator reagent that changes color or provides fluorescence ifamplification occurs, and wherein the method further comprises detectinga change in a signal that indicates the presence of coronavirus in thesaliva of the subject.

The method may include amplification reagents for LAMP as specified inthe kit above and single or multiple primer sets as specified abovealso. For example, the amplification reagents may include LAMP primersets targeting a second viral genome that is not a coronavirus whereinthe LAMP primer sets are combined in a single reaction mix and whereinthe LAMP primer set for the coronavirus is linked to a colorimetricindicator that changes color after amplification that is detectable atone wavelength and a second LAMP primer set for amplifying a second noncoronavirus nucleic acid, having a colorimetric or fluorescent indicatorthat changes color after amplification that is detectable at a secondwavelength.

Although the above examples of methods may include a sequencing stepafter the amplifying step.

One embodiment, provides for a method for amplifying a target nucleicacid by LAMP, that includes: (a) combining in a mixture, a biologicalsample from a mammalian subject with a buffer comprising a poloxamer;(b) heating the mixture to a temperature of at least 65° C. for aneffective time to denature proteins in the biological sample; (c)allowing the sample to cool; and (d) amplifying one or more nucleicacids from the mix by LAMP where the biological sample may be saliva, anasal swab or a buccal swab.

In one embodiment, a kit is provided for use in diagnostic detection ofa target nucleic acid and variants thereof having undefined mutations,obtained from a cell or virus in a biological sample. The kit mayinclude (a) a lyophilized mixture of a strand displacing polymerase andan indicator reagent and optionally a lyophilized reverse transcriptase,wherein (i) the indicator reagent is characterized by its ability tochange color or provide fluorescence in a nucleic acid amplificationreaction; and (ii) the strand displacing polymerase when rehydrated iscapable of amplifying DNA at a temperature in the range of 50° C.-68°C.; and (b) a universal primer set suitable for loop mediatedamplification (LAMP) of the target nucleic acids and variants thereofcontaining undefined mutations within one or more of the primer bindingsites. Any of the reagents in the kit may be combined in a mixture in asingle container or provided in separate containers.

The universal primer set as defined below is preferably suitable forLAMP and is capable of hybridizing to the target DNA in the presence ofa plurality of undefined mutations to provide a positive result for thetarget DNA in a predetermined assay time period otherwise determined fora positive sample of a target nucleic acid having a known sequence. Forexample, the universal primer set may be similarly diagnostic for thetarget nucleic acids and variants thereof where deletions and additionsin the BIF and FIP primer binding sites of the variants do not exceed6-9 nucleotides. In one example, the target DNA is the reversetranscription product of an RNA virus, for example, a coronavirus. Inone example, the indicator reagent is a molecular beacon.

The kit may include (c) lysis reagent in a container for receiving thebiological sample, wherein the lysis reagents comprise a reducing agentand a metal chelator. The lysis reagent may include the reducing agentis Tris (2-carboxyethyl) phosphine hydrochloride (TCEP). The lysisreagents may include at least one of a salt of C—(NH₂)₂NH⁺ and apoloxamer.

The kit may include one or more of components in (a)-(b) are immobilizedon a substrate.

The lyophilized indicator reagent may be a metallochromic dye or amolecular beacon. If a reverse transcriptase is included in the kit, thereverse transcriptase may be a virus encoded reverse transcriptase, or abacteria encoded intron II reverse transcriptase.

In one embodiment, a method is provided for detecting a target nucleicacid or unknown variant thereof in a biological sample by LAMP, thatincludes combining the biological sample with a lysis reagent to form alysis mix; incubating the lysis mix at a temperature of at least 60° C.for a period of time in the range of 2 minutes to 45 minutes; combiningin a reaction mix, an aliquot of the heat treated lysis mix withamplification reagents comprising a strand displacing polymerase, areversible inhibitor of the polymerase, nucleoside triphosphates, and atleast one set of LAMP primers that is capable of hybridizing to thetarget nucleic acid and to undefined variants of the target nucleicacid; and incubating the reaction mix for a reaction positive period oftime under amplification conditions for LAMP to detect the presence ofthe target nucleic acid or undefined variants thereof in the sample. Anadditional step may be included of sequencing the target nucleic aciddetected in the method to determine whether it is a variant of thetarget nucleic acid and characterizing any mutations.

The lysis reagent in the method may include a reducing agent and a metalchelating reagent. Once combined with the biological sample, the lysismix may be heated to 95° C. for 5 minutes.

The method may include a reverse transcriptase and a reversibleinhibitor of the reverse transcriptase in the amplification reagents.Any of the amplification reagents and/or lysis reagents may belyophilized prior to combining with the biological sample. Any of theamplification reagents and/or lysis reagents may be immobilized on amatrix prior to or during the method.

An example of a biological sample is saliva for detecting a targetnucleic acid such as an RNA virus for example, a coronavirus is saliva.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the office upon request and paymentof the necessary fee.

Figures that show results from pH dependent colorimetric LAMP rely on acolor change from red/pink (negative) to yellow (positive). These colorsare represented in the figures by replacing yellow (positive) in thetubes and on plates by black and by replacing red/pink (negative) withhash lines against a white background. FIGS. 11A, 13A-13D, and 14describe color changes from metallochromic dyes where colors change frombrown/orange to yellow. These colors are represented by dots that varyin density according to whether the reaction is positive (high density)or negative (1 or 2 dots) or somewhere in between.

FIG. 1 shows detection sensitivity of synthetic SARS-CoV-2 RNA ampliconsby a pH dependent colorimetric LAMP assay using phenol red as theindicator that changed color from red to yellow when amplification of atarget nucleic acid occurred, causing the pH of the reaction mixture todecrease. 5 sets of LAMP primers (1a-A, 1a-B, N-A, N-B, and 1a-C) weretested with templates ranging from 120×10⁶ to 120 copies of viral RNA(Twist Biosciences), or a no-template control (NTC). Yellow, positiveamplification; pink, no amplification.

FIG. 2A—FIG. 2B shows that RNA can be detected as efficiently as DNAusing pH dependent colorimetric LAMP (New England Biolabs, Ipswich,Mass. (M1800)) using the primers described in Example 1 that targetedtwo different SARS-CoV-2 sequences.

A comparison of RNA and gBlock double-stranded DNA (dsDNA) templates inLAMP amplification is shown using real time amplification curves. Twoprimer sets (ORF1a-A in FIG. 2A and Gene N-A in FIG. 2B) were used toamplify either RNA (green curves, dilutions from 120×10⁶ to 120 copies)or gDNA (blue curves, 60×10⁶ to 60 copies). For ORF1a-A primer set, thegBlock is faster than RNA template; for Gene N-A primer set, the RNA isslightly faster. Each “cycle” represents 20 seconds, with 30 minutetimepoint noted by dashed line.

FIG. 3A and FIG. 3B shows that pH dependent colorimetric LAMP can detectviral genomes in total cell lysates and whole blood without requiring apurification step to remove total RNA. The primer sets described inExample 1 were used here. Lysis was performed using a cell lysis reagent(Luna® Cell Ready (New England Biolabs, Ipswich, Mass.)).

FIG. 3A shows direct RNA LAMP detection using total cell lysate. Theapproximate maximum number of copies of synthetic RNA added to each LAMPreaction is shown. The viral RNA was spiked into a Hela cell lysate. For4800 copies of viral RNA, there were about 200 Hela cells present. NC,no cell, and no template control.

FIG. 3B shows colorimetric LAMP detection of various amounts of targetRNA spiked in whole blood. The number of copies of the target RNA thatcould be detected are shown. In a control, 5 ng of Jurkat total RNA wasadded, which is similar to the total RNA present in the reaction withblood samples.

FIG. 4A—FIG. 4B shows the detection of various closely related nematodeparasites was achieved for as little as 0.01 pg of nematode DNA in anenvironmental sample using the colorimetric LAMP described in Example 1.The control was a reagent mix absent nematode sample. Although Example 1describes the test for an RNA virus, the same methodology applies todetecting the DNA from the various nematodes using an appropriate set ofprimers. As indicated in the figure, the assay could detect 0.01 pg-0.1pg of nematode parasite DNA.

FIG. 5A-FIG. 5B shows that pH colorimetric LAMP is a useful diagnostictool for detecting tick borne pathogens as it is both sensitive andspecific for the target.

FIG. 5A shows detection of 1.28 fg target DNA from a specific targettick borne pathogen, with negative results (pink) from samples ofnon-target tick borne pathogens and hosts.

FIG. 5B shows specificity for the target DNA with negative (pink)results for non-target DNA. 1) no template DNA control; 2) pathogentarget DNA; 3) DNA from other tick-borne pathogen 1 (negative control);4) DNA from other tick-borne pathogen 2 (negative control); 5) DNA fromother tick-borne pathogen 3 (negative control); 6) tick DNA; 7) mosquitoDNA; 8) DNA from other tick-borne pathogen 4 (negative control).

FIG. 6A shows that pH-dependent colorimetric LAMP sensitivity isunaffected by the presence of dUTP and UDG. The endpoint color changewith target nucleic acid in two Carryover Prevention WarmStart®Colorimetric LAMP 2× master mixes (abbreviated CP-LAMP MM) (New EnglandBiolabs, Ipswich, Mass.). Two CP-LAMP MM contained a 50/50 mixture ofdUTP/dTTP replacing dTTP. One CP-LAMP MM did not contain UDG (no UDG).One CP-LAMP MM (with UDG) includes 0.02 U/μL thermolabile uracil DNAglycosylase (UDG) (New England Biolabs, Ipswich, Mass.). A third LAMP MMcontained neither UDG nor dUTP. In all other respects the same protocolswere followed as described in Example 1 and in the figures above.

FIG. 6B shows that carryover of polynucleotide substrate is effectivelyprevented by including dUTP in the LAMP reaction prevented over 10 folddilutions of the dU template from a first sample to a second master mixthat does not contain any target nucleic acid. Each sample from left toright is a 10 fold dilution of the previous sample. At 50 fold-60 folddilution, the carryover material was destroyed by the thermolabile UDG(0.02 U/μl). Carryover prevention (CP) was determined in CP-LAMP MM.

FIG. 7 shows that lyophilized pH-dependent LAMP MM is equally effectivewhen compared to non-lyophilized LAMP MM stored at −20° C. The startingpH of the lyophilized LAMP when reconstituted was reduced by 0.25 unitsin this example.

FIG. 8A-FIG. 8C shows real time detection of target RNA (Jurkat totalRNA) using a lyophilized LAMP MM and an HMBS2 primer set and a −20° C.storage preparation of LAMP MM. The LAMP MM contained the fluorescentdye (Syto-9) for following amplification. FIG. 8D provides a comparisonof the rate of LAMP using a previously lyophilized LAMP MM or a MMstored at −20° C.

FIG. 9A-FIG. 9C shows that no difference in sensitivity of the LAMPreaction was observed using a LAMP MM versus −20° C. stored LAMP MM forDNA and RNA analyses, where RNA analysis additionally required a reversetranscriptase in the MM. FIGS. 9A and 9B showed a color change withphenol red while FIG. 9C used a fluorescent dye to detect amplification.WarmStart® LAMP Kit (DNA & RNA) is provided New England Biolabs.

FIG. 10 shows the mechanism of the colorimetric response for4-(2-pyridylazo) resorcinol (PAR). PAR is a known metallochromicindicator which, in the absence of metals in solution, exhibits a yellowcolor. When complexed with manganese in solution PAR produces a redcolor. During the nucleic acid amplification process, pyrophosphate isproduced as a by-product of primer nucleic acid polymerization. Byincluding a small amount of manganese ions in an amplification reaction,PAR is initially complexed with the metal and therefore in a red-coloredstate. The pyrophosphate by-product sequesters manganese with a higheraffinity than does PAR, resulting in the dissociation of Mn from PAR andthereby returning PAR to a yellow-colored state.

FIG. 11A and FIG. 11B shows that because pyrophosphate exhibits a higheraffinity for manganese ions than PAR, this property can be used todetect amplification of nucleic acids using LAMP. Pyrophosphategenerated during nucleic acid polymerization precipitates manganese fromsolution, thus disrupting the PAR:Mn complex and restoring the yellowcolor. This is demonstrated spectroscopically by spiking inpyrophosphate to restore the yellow color (bottom row) (FIG. 11A).

The color change can be further enhanced by the addition of Triton X-100(FIG. 11B).

FIG. 12A shows that PAR has been demonstrated to be compatible with usein microfluidic paper-based analytical devices (μPADs) (Meredith, et al.Anal. Methods (2017) 9, 534-540). FIG. 12B shows the results of apaper-based spot test showing metal-PAR reactivity (orange and red colorformation) for a number of transition, alkali, and alkaline earthmetals.

FIG. 13A-13D shows that PAR provides the means of a colorimetricendpoint for LAMP in a LAMP MM. A color change is observed whenprecipitation of manganese occurs that is caused by the release ofpyrophosphates in a LAMP reaction. The enzymes added to perform LAMP inthe presence of resorcinol and conditions of the reaction are asfollows:

FIG. 13A: DNA polymerase Bst LF: (M0275) in 1× ThermoPol® added to 1 ulof Hela Cell gDNA; FIG. 13B: Bst 3.0: (M0374) in 1× IsothermalAmplification Buffer II absent gDNA; FIG. 13C: Bst 2.0: (M0537) in 1×Isothermal Amplification Buffer plus lug gDNA; and FIG. 13D: WarmStartBst 2.0: (M0538) in 1× Isothermal Amplification Buffer (New EnglandBiolabs, Ipswich, Mass.);

PAR concentrations left to right: 150 μM, 100 μM, 75 μM, 50 μM;

Mn²⁺ concentration: 0.5 mM MnCl₂;

LAMP Reaction Incubation: 65° C. for 1 hour;

LAMP Primer Set: BRCA2b FIP/BIP/F3/B3/LF/LB.

Target polynucleotide is the BRCA gene in Hela cell genomic DNA.

The non-template control retains the red color of PAR bound to manganeseions while the positive sample turned yellow corresponding to thereaction of manganese with pyrophosphate.

FIG. 14 shows the strong color reaction of a PAR-based LAMP in thepresence of 2% Triton X-100 and 0.5 mM MnCl₂, Bst 2.0 polymerase, 1×Isothermal Amplification Buffer, 200 μM PAR and the BRCA2b primer setusing 1 ul Hela cells. The non-template control retains the red color ofPAR bound to manganese ions while the positive sample turned yellowcorresponding to the reaction of manganese with pyrophosphate.

FIG. 15 shows examples of 4 different guanidine salts (also calledguanidinium salts) for enhancing the LAMP reaction.

FIG. 16 shows that guanidine hydrochloride (GuCl) increases the reactionspeed of a LAMP amplification reaction. Detection of two differentgenes-BRACA and CFTR were achieved using LAMP MM and increasingconcentrations of GuCl.

FIG. 17A-17C shows that GuHcl was effective in increasing the rate ofisothermal amplification reactions and this effect was enhanced byselecting a range of concentrations for NaCl or KCl in the reactionbuffer.

FIG. 17A shows the results of standard HDA reactions in IsoAmp II kit(H0110) and 0.1 ng plasmid, with 10 mM NaCl versus 40 mM NaCl, in whichguanidinium hydrochloride was added at a final concentration of 0 mM-60mM. The reactions were performed at 65° C. and EvaGreen® dye (Biotium,Inc., Hayward, Calif.) was included to monitor the progression ofamplification. The effect of reducing NaCl concentrations was mostnoticeable at higher concentrations of GuCl (30 mM-60 mM guanidinehydrochloride) resulting in a reduction of Time to threshold (Tt) of 35minutes (40 mM NaCl) to 12.3 minutes (10 mM NaCl).

FIG. 17B shows an increase in the rate of amplification using the LAMPassay described in Example 1 and Bst 2.0 DNA polymerase with a lambda1primer set and 0.5 ng lambda DNA in ThermoPol buffer containing 10 mMKCl plus or minus 30 mM guanidine hydrochloride. The addition ofguanidine stimulated the LAMP amplification rate significantly at thelower end of the KCl concentration (less than 40 mM KCl).

FIG. 17C shows an increase in the rate of amplification using the LAMPassay described in Example 1 and Bst 3.0 DNA polymerase (also referredto as BTB3) with a lambda1 primer set and 0.5 ng lambda DNA in anisothermal amplification buffer containing 50 mM KCL plus or minus 30 mMguanidine hydrochloride. The addition of guanidine stimulated the LAMPamplification rate significantly at the lower end of the KClconcentration (less than 100 mM KCl).

FIG. 18A-18D shows that guanidine hydrochloride not only increases LAMPreaction speed but also improves the limit of detection sensitivity.

FIG. 18A shows that colorimetric LAMP could detect 100 copies ofsynthetic SARS-CoV-2 RNA with 40 mM guanidine hydrochloride using primerset 1. The color change from pink to yellow indicates a positivedetection. “None” denotes no guanidine hydrochloride. In the presence of40 mM guanidine hydrochloride, 8/8 positive reactions were detected,whereas 5/8 positive reactions were detected without it.

FIG. 18B shows the results of real time colorimetric LAMP using primerset 1 in the presence and absence of 40 mM Guanidine HCl. The reactionalso contains 1 μM dsDNA binding dye Syto-9 for monitoring the real timeprogression of the amplification.

FIG. 18C shows the results with 4 different primer sets. In all cases,sensitivity was increased in the presence of guanidine hydrochloride.The percentage of positive reactions for detecting 100 copies ofSARS-CoV-2 RNA is shown in the table. The table shows that guanidineimproves the detection sensitivity of all primer sets.

FIG. 18D shows a diagram of SARS-CoV-2 with the location of 2 templatesequences (E and N) in the target nucleic acid.

FIG. 19A-19C shows that guanidine allows efficient multiplexing of LAMPamplifications with multiple LAMP primer sets in the same reactionwithout adverse effects on the rate of amplification while significantimprovements in sensitivity were observed.

FIG. 19A shows sensitivity of pH colorimetric LAMP by the percentage ofpositive samples detected using single sets of primers (identified assets 3 and 4) and when sets 3 and 4 are combined in the presence orabsence of 40 mM guanidine hydrochloride in a 40 minute incubation. Thetables shows that 92.2% positives were detected for known test samplescontaining 50 copies synthetic SARS-CoV-2 RNA using a combined set ofprimers 3 and 4 with guanidine, compared with 28% for single sets ofprimers in the absence of guanidine.

FIG. 19B shows the sensitivity of pH colorimetric LAMP for detecting12.5 copies of synthetic SARS-CoV-2 RNA in the presence of guanidiniumhydrochloride and a plurality of primer sets. The results shows anincrease of detection rate with any combinations of 2 primer sets (3+4,3+5, 4+5). The reactions including all 3 primer sets (3+4+5) also showedfurther increase of detection rate over any 2 primer sets, providingdetection of 57% of all positives in a 40 minute incubation. Thereactions without template remained negative and showed no sign ofamplification signal, indicating robust specific amplification.

FIG. 19C shows that real time amplification with guanidine hydrochlorideresulted in an expected rate of amplification with a combination ofprimer sets 3 and 4 and 50 copies of template CV-19 RNA where thecombination of primer sets did not adversely affect the rate ofamplification.

FIG. 20 shows an example of the use of lysis buffer for SARS-CoV-2detection in saliva where 900 ul of saliva from a patient is added to100 ul of 10× lysis buffer (1), mixed (2) and then heated to 95° C. for5 minutes (3). 2 ul of the sample is then added to 18 ul of pHcolorimetric LAMP Master mix either containing primers to SARS-CoV-2(target) or primers for actin (control) (4). After an incubation for 45minutes at 65° C., the test tubes were examined for a color change frompink to yellow indicative of the presence of SARS-CoV-2 (5).

FIG. 21A-21B shows that various ratios of the reagents in the lysisbuffer spiked with synthetic SARS-CoV-2 RNA were tested to determinewhich combination if any interfered with pH colorimetric LAMP and if notwhich conditions provided the greatest sensitivity for detecting 40copies of the virus genome. The results did not suggest any interferenceand the saliva lysis mixture containing 4 mM TCEP (reducing agent) and75 mM LiCl at pH 8.0 with 400 mM guanidine hydrochloride (GnHCL) gavethe best results.

FIG. 21A shows 6 different conditions for the saliva lysis buffer.

FIG. 21B shows the color changes indicative of a positive result underthe 6 different test conditions where 4 mM TCEP (reducing agent) and 75mM LiCl at pH 8.0 with 400 mM guanidine hydrochloride (GnHCL) resultedin 100% detection of 40 copies of viral genome.

FIG. 22 shows that 8 mM TCEP in addition to 75 mM LiCl and 400 mM GnHCLperformed similarly to 4 mM TCEP in the saliva lysis buffer when 5 μl10× lysis buffer was added to 45 μl saliva sample containing inactivevirus particles (SeraCare) and heated for 5 minutes at 95° C. 2 μl ofthis sample was then added to 18 μl LAMP master mix and incubated for 35minutes at 65° C.

FIG. 23A-23D shows the effect of varying the LAMP assay time after the 5minute saliva lysis reactions on saliva spiked with 10,000 cps/mlsynthetic SARS-CoV-2 RNA (20 copies/2 μl) using a saliva lysis buffercontaining 8 mM TCEP, 0 mM LiCl/75 mM LiCl and 400 mM GnCL. Increasedsensitivity was observed over time with the presence of LiClconsistently contributing to increased sensitivity as the time ofincubation increased beyond 35 minutes. Saliva not containing RNA andH₂O were used as negative controls.

FIG. 24 shows that when a saliva sample spiked with a known copy numberof synthetic SARS-CoV-2 input (Twist) is treated with saliva lysisbuffer (8 mM TCEP, 75 mM LiCl and 400 mM GnCl), followed by RT-qPCR, thelysis buffer was shown to have minimal or no adverse effect on theRT-qPCR reaction.

FIG. 25 shows that the lysis buffer described in FIG. 23A-23D and FIG.24 can provide similar sensitivity of virus detection as that reportedby others after purification of the viral RNA from nasopharyngeal swabsand saliva (Wyllie et al. MedRxiv Apr. 22, 2020:https://doi.org/10.1101/2020.04.16.20067835).

All positive nasopharyngeal swabs (n=46) and saliva samples (n=39) werecompared by a Mann-Whitney test (p<0.05). Bars represent the median and95% Cl. Our assay detection limits for SARS-CoV-2 using the US CDC “N1”assay is at cycle threshold 38, which corresponds to 5,610 viruscopies/mL of sample (shown as dotted line and grey area).

FIG. 26 shows that endpoint absorbance ratio (432 nm/560 nm wavelengths)at a range of pH from pH 4-pH 11 can be measured by a colorimeter. Thehighest positive signal at 560 nm is between pH 9-pH 11 and the highestnegative signals at 432 nm occurs at pH 4-pH 6. The 432/560 nm signalratio can be used to determine positive and negative samples inpH-dependent colorimetric LAMP.

FIG. 27 shows the absorbance results after pH dependent colorimetricLAMP was performed on samples containing synthetic SARS-CoV-2 RNA usingdual primer sets of N2 and E1 after no incubation at 65° C. and after 20minutes incubation at 65° C. The samples were allowed to cool to roomtemperature before the color was measured in the SpectraMax® (MolecularDevices, San Jose, Calif.). Data is provided from a SpectraMax readoutthat provided 100% detection of 20 copies of SARS-CoV-2 RNA and 62%detection of 10 copies of SARS-CoV-2 RNA using a sample spiked with 20copies of SARS-CoV-2 RNA.

FIG. 28A-28F shows an example of an automated workflow that permits100,000 reactions in about 20 hours. This is calculated from a batchsize of 5,760 reactions (15×384 well plates or 60×96 tube racks) with aprocess time of 40 minutes/sample and 100 minutes/batch.

FIG. 28A shows how an individual saliva sample from a collection tubemight be placed in a tube containing saliva lysis buffer in a 96 tuberack.

FIG. 28B shows a robot that can transfer samples (for example 3 μl) fromindividually 2D barcoded sample collection tubes or batches of 4×96 tuberacks to 384 well plates with a linear barcode to associate each sampleto a discrete well location in 4 minutes.

FIG. 28C shows a robot liquid handler that can add for example 17 μl ofreaction mix (e.g. 2 μL 10× primer mix, 10 μL WarmStart ColorimetricLamp 2× Master Mix (M1800), 5 μL of DNAse, RNAse free H2O) into the 384well plate within about 1 minute.

FIG. 28D shows a stack of plates each with plastic seal ready for theLAMP reaction.

FIG. 28E shows two devices for performing LAMP that requires incubationat 65° C. for a period of time such as 30 minutes. This may be achievedby means of a horizontal conveyor belt that sends each plate through aheated chamber so that the residence time in the chamber is the desiredincubation time. Alternatively, this may be achieved by stacking platesin a tower incubator where heating occurs for the programmed time.

FIG. 28F shows a robotic plate handler that takes the 384 plates fromthe incubator and places them in sequence in a SpectraMax or otherspectrophotometer (absorbance reader) that records the 2D barcode on theplate and the color of each well at specific wavelengths.

FIG. 29 shows a schematic for handling large numbers of patients at apop-up laboratory using embodiments of the rapid LAMP method fordetecting SARS-CoV-2.

FIG. 30 (a)-(c) shows a workflow for a LAMP reaction using each of 3different types of sample collection. These are:

FIG. 30 (a) collect patient saliva sample into an empty tube,inactivation of any infectious virus in the absence of a buffer using atemperature of 65° C. for 30 minutes, 75° C. for 15 minutes or 95° C.for 5 minutes and then transfer for example, 1 volume of the sample into1 volume of 2× buffer or an equivalent ratio using 4× buffer or 10×buffer resulting in a 1× mixture;

FIG. 30 (b) incorporating a volume of a concentrated viral inactivationbuffer (for example 2×, 5× or 10×) into a compartment of a salivacollection tube prior to collection of saliva from the patient in thesame tube. After collecting the saliva and closing the lid of the tube,the sample is mixed with viral inactivation buffer released from thecompartment;

FIG. 30 (c) a volume of viral inactivation buffer is present in a sampletube for receiving a nasal or oral swab from a patient where thecontents of the swab are directly released into the buffer. In all casesin this example, the viral inactivation buffer contains a poloxamersurfactant such as PF68 suitable for reducing RNase activity in additionto a reducing agent (e.g., TCEP) and a metal chelator (e.g., EDTA).Also, in all three types of sample collection (a)-(c), a heating step at95° C. for 5 minutes is performed that is generally expected to break upthe cells and any viruses releasing RNA while inactivating nucleases. Analiquot of the patient sample is then transferred into standard LAMPmaster mix containing a DNA polymerase, and reversible inhibitor, areverse transcriptase with reversible inhibitor, primer sets andnucleoside triphosphates for fluorescent LAMP (e.g., fluorescent LAMPusing an intercalating dye or DARQ LAMP for multiplexing) orcolorimetric LAMP. The sensitivity of the colorimetric LAMP is improvedby the additional presence of guanidium salt. Alternatively, though notshown, an aliquot can be used for RT-qPCR or for any other diagnosticassay including sequencing.

FIG. 31A-31E show minimal interference between primer sets in amultiplex reaction. The duplex of the 5′-modified version of the FIPprimer (Q-FIP) annealed to Fd complementary sequence on the SARS-CoV-2target genome (50 copies) and the actin control in the presence of 1, 2,3 and 4 primer sets. 24 samples were tested for SARS-CoV-2 and 8 sampleswere tested for the actin control. Rox (Integrated DNA technologies(IDT) Wisconsin) was used for the fluorescent label for ACTB while JOE(IDT) was used for E1.

FIG. 31A shows DARQ LAMP with E1 and ACTB primer sets in the presence ofSARS-CoV-2 target RNA and ACTB target RNA. SARS-CoV-2 signal.

FIG. 31B shows DARQ LAMP with E1, ACTB and EIA primer sets (all withQ-FIP:Fd) in the presence of SARS-CoV-2 target RNA and ACTB RNA.

FIG. 31C shows DARQ LAMP with E1, ACTB, EIA and EIB primer sets (allwith Q-FIP:Fd) in the presence of SARS-CoV-2 target RNA and ACTB RNA.

FIG. 31D shows DARQ LAMP with E1, ACTB, EIA and EIB primer sets (allwith Q-FIP:Fd) in the presence of ACTB RNA.

FIG. 31E provides a summary table of results provided by 31A-31D.

FIG. 32A-32C shows that SARS-CoV-2 and Influenza strain A or B can bedetected in a single sample in a multiplex reaction.

FIG. 32A DARQ LAMP detection of SARS-CoV-2 (labeled with JOE) and Flu A(labeled with Cy5) provides similar results to the detection of Flu Aand SARS-CoV-2 alone.

FIG. 32B DARQ LAMP detection of SARS-CoV-2 (labeled with JOE) and Flu B(labeled with FAM) provides similar results to the detection of Flu Band SARS-CoV-2 alone.

FIG. 32C DARQ LAMP detection of Flu A (Cy5) and Flu B (FAM) providessimilar results to the detection of Flu A and Flu B alone.

FIG. 33 shows a supported lipid bilayer (SLB) 96 well plate withnumbered wells containing serial dilutions of virus spiked into negativesaliva samples.

FIG. 34. shows the results of a colorimetric LAMP assay usinginactivated SARS-Cov-2 virus spiked negative saliva. The dilution seriesshows samples where the limit of detection (LOD) is 40 cps/uL.

FIG. 35 shows that all samples were positive that contained 40 copies ofinactivated virus into saliva with LAMP reactions for each sample.

FIG. 36A-36F shows that mutation position effects on RT-LAMPamplification does not affect sensitivity but can make small changes intime to endpoint. Plots of the effects of change relative to the WTprimer set for three genes from SARS Covid 2-As1e (circle), E1 (square),N2 (triangle) are provided for each primer.

FIG. 36A—F3 primer; FIG. 36B—B3 primer; FIG. 36C—FIP primer; FIG.36D—BIP primer; FIG. 36E-Loop F primer; and FIG. 36F—Loop B primer.

FIG. 37A-37B shows that a molecular beacon assay is an effective endpoint for a universal LAMP assay.

FIG. 37A describes the design of SGF LAMP primers and beacons. Upperpanel shows the locations of various LAMP primers and molecular beacons.The lower panel compares the sequences for wt, SGF deletion, SGFwt-MBand SGFdel-M B. Dashes: bases deleted in SGF deletion; Bold: LNA base;Underlined: stem region; Italics, non-target sequence, attachedfluorophores and quenchers.

FIG. 37B shows comparable sensitivity of detection of SARS-CoV-2sequences including wild type (SGFwt) and variant RNA (SGFdel) by LAMPusing molecular beacons. Results of LAMP reactions with either WT RNA(left panels) or B.1.1.7 RNA (right) in the presence of SYTO-9,SGFdel-MB, or SGFwt-MB are shown. The primer set amplifies both the wtand B.1.1.7 RNA with similar efficiency as detected with SYTO-9 (top).When MB beacon was added as a reporter, both SGFdel-MB (middle) andSGFwt-MB (lower) showed only with their intended template RNAs from50-10,000 copies.

FIG. 38 shows the structure of a poloxamer.

DESCRIPTION OF EMBODIMENTS

Embodiments utilize isothermal amplification, such as LAMP, and simplevisual detection of amplification in a liquid or on a reaction matrixfor potential use in rapid, field applications. LAMP is an isothermalamplification protocol first developed by the Eiken Chemical Co. inJapan (see for example, Notomi, et al. Nucleic Acid Research (2000) 28,E63). LAMP is also described in detail in U.S. Pat. No. 6,410,278 andMori, et al. (J. Infect. Chemother. 2009 15: 62-9). In LAMP, fourprimers recognize six unique target sequences on the template strand.Two of the primers are designated as “inner primers” (FIP and BIP) andtwo are designated “outer primers” (F3 and B3). In addition tocontaining a sequence that is complementary to a target sequence attheir 3′ ends, the inner primers also contain a tail that comprises asequence that is downstream of the 3′ end of primers in the template.Thus, extension of an inner primer results in a product that has aself-complementary sequence at the 5′ end. Displacement of this productby an outer primer generates a product that has a loop at the 5′ end.Thus, the primer sets used in LAMP typically contain four, five or sixtemplate-complementary sequences, where four sequences are found at the3′ ends of the primers and two of the sequences are found at the 5′ endsof two of the primers. The initial reaction in LAMP results in a DNAproduct that has a dumbbell-like structure. In this product, the endsform stem loops and the single stranded region in between the stem loopsis copied from the template. This product self-primes its ownamplification to amplify the template sequence. LAMP uses astrand-displacing polymerase, and is isothermal, that is it does notrequire heating and cooling cycles.

Primer sets for LAMP as used herein refer to four to six primers thatincludes optionally loop forward and/or backward primers (LF and LB) inaddition to forward internal primers (FIP) and backward internal primers(BIP) and forward primer (F3) and backward primer (B3).

LAMP assays are described herein that enable rapid and sensitivedetection of target nucleic acids; such as a nucleic acid of orassociated with a pathogen, such as a virus, in a human or animalpopulation. These assays are simple and portable while retainingsensitivity and minimizing false positives and negatives. The LAMPassays described herein rely on detection of a change in some aspect,such as fluorescence, or color of a dye in a reaction mix due to achange in pH or metal ions. Turbidity may also be used as an end pointin some LAMP assays.

The LAMP assays described herein were tested for their ability to detectnot only the target nucleic acid but also variants thereof. The testsample was SARS-Cov-2 which has been able to evolve by mutating its RNAto escape the host immune system and to be more infectious. For example,SARS-CoV-2 variant strains B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma),B.1.617.2 (delta) and C.37 (lambda) emerged in different regions of theworld, and rapidly became the most frequently isolated strainsworldwide. They have numerous and distinctive missense mutations, mostprominently located in the spike protein. While only alpha carries theΔ69-70 deletion, all but delta carry a 9-base, 3-amino acid deletion atpositions 3675-3677 in the Orf1a sequence (termed the “SGF” deletion).

Here we established the first comprehensive screen of LAMP primertolerance to mutation, investigating a single base mutation at everyposition of every primer in three prominent SARS-CoV-2 RT-LAMP assays.Remarkably, we find very little impact of the single base changes, withonly marginal effect on speed in most positions. The robustness ofRT-LAMP to sequence variation is a significant benefit to its adoption,with reduced worry about deleterious effects from the commonly emergingsingle-base changes that could occur with some frequency in the regionstargeted by the LAMP primers. Additionally, many RT-LAMP assays combineprimer sets for added speed and sensitivity (Zhang et al. Biotechniques69(3), 178-185 (2020)) adding an additional layer of protection againstpossible sequence variation.

It is desirable to detect unknown variants in order to make anassessment of the change in the virus population and to monitor itsspread. Once a positive result is obtained, the virus can be sequencedand the mutations defined. After the mutations have been discovered, newprimer pairs can be used to detect viral infections with confidence bymeans of RT-PCR amplification tests.

This relatively large deletion provides a reliable means for designingmolecular diagnostics specific for these strains.

The effect of SARS-CoV-2 sequence mutations on RT-LAMP amplification wasanalyzed by creating 572 single point mutation “variants” covering everyposition of the LAMP primers in 3 SARS-CoV-2 assays and analyzing theireffects with over 4,500 RT-LAMP reactions. With this evaluation weremarkably observed only moderate effects on amplification speed and noeffect on detection sensitivity, highlighting RT-LAMP as an extremelyrobust technique for viral RNA detection. Additionally we describe theuse of molecular beacons to sensitively distinguish variant RNAsequence.

All abbreviated references herein to embodiments that relate to“colorimetric tests” refer to detection of a target nucleic acid (e.g.,a target pathogen nucleic acid) by a change in color of a dye in areaction mix due to a change in pH or metal ion binding or dissociation.

The “target nucleic acid” as used herein refers to any nucleic acid thatis targeted by primers to determine its presence in a biological sample.

The diagnostic LAMP methods described herein may be used for detectingpathogens, including any of: prokaryotes such as bacteria, eukaryoticpathogens such as multicellular parasites, fungi, single cell pathogenssuch as trypanosomes or yeasts, and mycoplasma; as well as for use ingenetic tests, such as for genetic diseases, and in personalizedmedicine, which may require SNP detection or gene analysis of the genomeof a subject or RNA analysis to determine gene expression profiles inresponse to an environmental or metabolic event. Other diagnostic usesinclude testing food for undesirable biological entities and monitoringenvironmental samples which may include biological material and are herereferred to as biological samples also. Many of the examples providedherein are directed to the SARS-CoV-2 RNA virus because of a sensitivediagnostic test is of paramount importance. However, the improvements insensitivity described here may be applied to any colorimetric orfluorescent LAMP based assay to detect a target nucleic acid and indeedmay find applications in non-LAMP assays also.

Described herein are modifications to isothermal amplificationdiagnostic assays (e.g. LAMP) to improve their sensitivity. Suchmodifications and improvements may include one or more of:

-   -   (a) sample collection in water or weak buffer;    -   (b) sensitive detection of a pathogen such as a virus from a        sample body fluid such as saliva;    -   (c) avoiding nucleic acid purification for assaying for the        target nucleic acid in the sample;    -   (d) addition of one or more nuclease inhibitors (e.g., RNase        and/or DNase inhibitors) to the sample (e.g., into a sample        receiving tube) to prevent loss of sample and thereby to        increase overall sensitivity of the assay;    -   (e) addition of thermolabile Proteinase K to the sample (e.g.,        into the sample tube) to facilitate the efficiency of the LAMP        reaction by removing unwanted protein, followed by heat        inactivation of the Proteinase K prior to subsequent isothermal        amplification (e.g., LAMP or RT-LAMP) reactions;    -   (f) inactivating pathogens and nucleases in a sample by heat        treatment prior to or at the same time as the addition of lysis        buffer;    -   (g) addition of dUTP and UDG (e.g., thermolabile UDG) into the        reaction mix to prevent sample carryover when large numbers of        samples are being handled where the thermolabile UDG is        inactivated at the temperature of the LAMP reaction that is        further coordinated with the release of inhibition of the strand        displacing polymerase to allow amplification of the target to        occur while at the same time permitting incorporation of dUTP        into the amplified target DNA;    -   (h) addition of various components to the reaction mix to reduce        background and enhance signal, such as a helicase (see for        example, U.S. Pat. No. 9,920,358), or a carboxamide (see for        example, U.S. Pat. No. 9,546,358);    -   (i) other adjustments to reduce background and enhance signal,        such as optimizing the ratio of probes (see for example, U.S.        Pat. No. 9,074,249); design of primers or probes (see for        example, U.S. Pat. No. 9,074,243), improvements in the strand        displacing polymerase for use in LAMP (see for example, U.S.        Pat. Nos. 9,157,073 and 9,127,258) addition of guanidine salts,        use of reversible inhibitors of the DNA polymerase and reverse        transcriptase in the and/or in reverse transcriptases for use in        LAMP (see for example, U.S. Pat. Nos. 9,920,305, 9,580,698 and        9,932,567);    -   (j) storage of concentrated master mix at room temperature in a        lyophilized form for uses that include ease of transport and        storage at the test site;    -   (k) substituting a pH sensitive dye and associated amplification        buffer (of no greater than 5 mM Tris) with a metallochromic dye        such as resorcinol (PAR), manganese ions, a detergent such as a        non-ionic detergent and a standard isothermal reaction buffer;    -   (l) storing lyophilized master mix with probes and        metallochromic dye or fluorescent dye on a paper, microfluidic        device, or polymer surface for use in a target specific strip        test where a liquid sample is added to the paper, microfluidic        device or polymer surface containing the lyophilized reagents        and a readout is obtained;    -   (m) adding a guanidine salt to increase the rate of the        isothermal amplification (e.g., LAMP) reaction and increase        sensitivity;    -   (n) using multiple sets of LAMP primers for use in amplifying a        single template to enhance sensitivity;    -   (o) using multiple sets of LAMP primers for amplifying multiple        target nucleic acids    -   (p) reducing the concentration of NaCl or KCl in the buffer in        the presence of guanidine salts to enhance the rate of reaction        and/or improve sensitivity of the LAMP assay;    -   (q) using a sample lysis buffer for direct analysis of nucleic        acids from a sample without purification where the lysis buffer        contains a guanidine salt and a reducing agent such as TCEP and        optionally LiCl and optionally a detergent or a poloxamer;    -   (r) use of a dual wavelength spectrophotometer to distinguish        positive from negative samples rapidly in high throughput        workflows and/or real time analysis;    -   (s) use of a high throughput automated workflow from sample        collection to recording of results to achieve at least 100,000        samples in 20 hours;    -   (t) a point of care kit that provides a positive/negative result        concerning the presence of a nucleic acid within 45 minutes of        receiving a sample and without instrumentation outside a source        of time- and/or thermostat-regulated heat;    -   (u) Improving sensitivity of the diagnostic test for target        nucleic acids contained in biological samples without requiring        isolation or purification of the nucleic acid;    -   (v) Endpoint detection of a positive results using visual color        change (colorimetric) that may be pH dependent or a change in        the color of a transition metal or by means of fluorescence such        as DARQ (Zhang et al. Biotechniques 70(3), 167-174 (2021));        molecular beacons (Sherrill-Mix et al. Genome Biol 22(1), 169        (2021)) or coupled to secondary molecular analysis platforms        such as CRISPR (Broughton et al. Nat Biotechnol 38(7), 870-874        (2020); and Jounget al. N Engl J Med 383(15), 1492-1494 (2020);        next generation sequencing (LamPORE and LAMP-Seq) (Ludwig et al.        Nat Biotechnol doi:10.1038/s41587-021-00966-9 (2021)); Peto et        al. J Clin Microbiol 59(6), (2021).

The combination of a quick sample preparation method with an easydetection process provides portable, field detection in addition to arapid screening for point-of-need testing applications. The use of thisdiagnostic methodology for a virus that represents an emergingsignificant public health concern provides applications outside oftraditional laboratories that will enable greater prevention andsurveillance approaches. These embodiments provide the basis for a modelfor inevitable future outbreaks of viral pathogens and indeed anyinfectious agent to dramatically expand the reach of testingcapabilities for better healthcare outcomes.

The term “Bst polymerase” refers to any of Bst large fragment or mutantof the Bst polymerase or Bst large fragment. Examples of mutants of Bstpolymerase are described in U.S. Pat. Nos. 9,157,073, 9,993,298, and9,127,258.

The term “master mix” refers to a combination of reagents which can beadded to a sample to execute a reaction in an assay where thecombination enhances the efficiency and speed of performing the assay.The master mixes described herein include a mesophilic strand displacingDNA polymerase and may additionally include other enzymes such as areverse transcriptase, uracil deglycosylase (also referred to herein asuracil DNA glycosylase) for example, a thermolabile UDG which becomesinactivated in the temperature range of 50° C.-60° C. or at 65° C.; anda thermolabile Proteinase K such as a thermolabile Proteinase K. Themaster mix may also include a reversible inhibitor of DNA polymeraseactivity. An example of a reversible inhibitor is an oligonucleotideknown as an aptamer that binds to the DNA polymerase and blocks itsactivity below a selected temperature (for example 50° C., 55° C. or 60°C.) but above that temperature, the oligonucleotide is disassociatedfrom the enzyme, permitting the reverse transcriptase to become active.In some embodiments, the master mix includes a reverse transcriptase anda reversible inhibitor of reverse transcriptase activity for inhibitingthe activity of these enzymes below 40° C. or below 45° C. or below 50°C. An example of a reversible inhibitor is an oligonucleotide known asan aptamer that binds to the reverse transcriptase and blocks itsactivity below a selected temperature (for example 40° C.) but abovethat temperature, the oligonucleotide is disassociated from the enzyme,permitting the reverse transcriptase to become active. This permitssetting up a reaction at room temperature while avoiding nonspecificamplification. The master mix may also contain inhibitors of nucleasessuch as RNase inhibitors and/or DNAse inhibitors. These inhibitors maybe chemical reagents such as poloxamers, and/or aptamers. However,nucleases may also be inactivated by submitting the sample combined witha lysis buffer to a high temperature for an effective time such as Themaster mixed may also include dNTPs such as dTTP, dATP, dGTP and dCTP aswell as dUTP for carryover prevention. For example, a 2× master mix maycontain the dNTPs in equal quantities except the dUTP at 50%concentration of the other dNTPs. The master mix may include singlestrand binding proteins and/or helicases to reduce nonspecificamplification. The master mix may include a pH-sensitive dye or ametallochromic dye. The master mix may be lyophilized or freeze dried.It may be preserved for storage in a suitable buffer that may contain atleast one reducing agent and at least one detergent and capable ofstorage at −20° C. for an extended period of time (for example months).The inclusion of a reducing agent is desirable if RNA is the templatenucleic acid. It is not required for DNA. The master mix for use in pHcolorimetric LAMP may have a low buffer concentration such as 5 mM Trisor less. A low buffer concentration is not required if a metallochromicdye of fluorescent dye is used to detect amplification. The master mixmay be prepared in a 2×, 3×, 4×, 5×, 10× or any suitable concentration.The master mix once diluted by the sample will result in a 1×concentration. The master mix may contain primers or primers are notcontained in the master mix.

Kits refer to a combination of materials that are needed to perform areaction. A kit may contain multiple tubes or a single tube. The kit mayinclude a mixture of lyophilized reagents and reagents in a storagebuffer. In one embodiment, the kits described herein contain multipletubes wherein the master mix is contained in one tube, guanidine salt ina second tube and oligonucleotide primers in a third tube. Inembodiments of the kit, unless the primers are lyophilized together withthe master mix, the oligonucleotide primers in the third tube comprise aplurality of sets of LAMP primers, for example two sets of primersconsisting of 8-12 primers where each set has 4-6 primers and whereinboth sets of primers target different sequences in a single nucleic acidor different nucleic acid targets. The third tube may contain more than2 sets of LAMP primers where a plurality of sets may target a singlenucleic acid or multiple different nucleic acid sequences. For example,a plurality of sets of LAMP primers in the third tube may target thegenome of SARS-CoV-2 and a different set or plurality of sets of LAMPprimers in the third tube may target the genome of influenza virus. Theplurality of LAMP primers in the third tube may additionally contain asample identification sequence if multiple samples are pooled forcombining with the kit. In one embodiment, the kit contains a 2× mastermix, a 5×-25× guanidinium salts, and 5×-25× of LAMP primer sets.

Sampling to Obtain Target Nucleic Acid

In embodiments, purified nucleic acid such as RNA or DNA, or directtissue or cell lysate can be analyzed in a colorimetric LAMP such as apH dependent colorimetric LAMP assay, a fluorescent LAMP assay or ametallochromic based LAMP assay. Either purified or lysate can be usedin a simple, rapid method for SARS-CoV-2 RNA detection.

In one embodiment, a sample is obtained from the nasal passages of apatient (human or mammal) by nasal swab or from the buccal cavity. Thebiological sample may be one or more of the following body fluids:saliva, sputum, mucous, blood, semen, urine, sweat, lymph fluid, feces.The biological sample may alternatively be a tissue sample. Thebiological sample may include pathogens such as parasites such asviruses, bacteria, archaea, worms, ticks, single cell organisms, andfungi, Samples may also be obtained from an environmental source such asa food, plant, sewage, water, dust, or surface swab of an object.Samples can be placed into a small volume of water saline, TE, orsuitable transport medium (for example, a universal transport medium forviruses) or directly into a lysis buffer that inactivates and breaksopen the pathogen if that is the diagnostic target while protecting thereleased target nucleic acids from nuclease digestion. The samples maybe further purified from lysis buffer or added without furtherpurification to a LAMP amplification master mix for testing for thepresence of a pathogen, gene, SNP, mutation or other nucleic acidtarget.

The pH of the sample may determine the type of assay detection that ispreferably used. For example, urine has an acid pH so a metallochromicbased assay that is not pH sensitive might be used, instead of a pHendpoint.

Saliva

The nucleic acids in saliva may come from cheek cells and tongue cellsas well as any typical oral microbial species. Saliva can be collectedfrom a human subject using a commercial collection device such asprovided by for example any of Boca Scientific Inc. (Westwood, Mass.),Salimetrics LLC (Carlsbad, Calif.), Mantacc (Shenzhen, China), GreinerBio-One BD Sputum Collection (Monroe, N.C.). Saliva has some challengesas a body fluid for nucleic acid analysis. Its pH and composition mayvary according to the biology of the individual and also according tothe recent intake of food and/or liquid into the mouth. For example, itis well known that a glass of water containing the juice of a squeezedlemon will cause the saliva to become acidic for a short time afterintake. Where the saliva based diagnostic test relies on anamplification procedure that is pH dependent such as pH dependentcolorimetric LAMP, the pH can be normalized to the extent necessary withrespect to subsequent dilution before initiation of the amplificationreaction so that the concentration of buffer in the final reactionmixture is less than a corresponding amount of 5 mM Tris whileoptimizing the pH to be preferably within the range of pH 7.9-pH 8.3.Alternatively, the subject providing the saliva might avoid ingestion ofa particular food or drink for a predetermined number of minutes (suchas 30 minutes) before providing the saliva sample.

Where saliva is tested for a pathogenic virus, it may be desirable toimmediately inactivate the virus in the receiving tube. In thesecircumstances, a detergent may be added to the collection tube ifstorage before analysis is intended. For example, Triton X-100 inrelatively high concentrations has been shown not to interfere with asubsequent colorimetric LAMP reaction (see for example, FIG. 11B). Thelysis buffer as described below will inactivate the virus and releasenucleic acid for amplification or for sequencing. The lysis bufferdescribed in Example 9 is suited for amplification generally asillustrated for RT-QPCR and for LAMP. The saliva lysis buffer may alsobe applied to other body fluids for a similar purpose. The lysis buffermay be used for obtaining nucleic acid samples suitable for directsequencing such as by Oxford Nanopore.

In one embodiment, the sensitivity of the methods described herein wastested in samples of human SARS-CoV-2 negative saliva spiked with eitherSARS-CoV-2 viral RNA (Twist Synthetic SARS-CoV-2 RNA Control 2(MN908947.3) (Twist Biosciences, San Francisco, Calif.) or virusparticles provided by SeraCare (Milford, Mass.). An aliquot of thespiked saliva samples was added to the lysis buffer that was thenanalyzed using a pH colorimetric LAMP assay as shown in FIG. 20. Usingthe methods described herein it was possible to detect less than a 100copies of viral RNA (80 copies of virus) derived from a saliva sample,more particularly less than 80 particles, more particularly, 40particles or less with up to 100% efficacy. This corresponded to lessthan 50,000/ml virus particles in the original sample where less than100 copies of virus were detected, less than 40,000/ml copies of virusin the sample (80 copies), 20,000 copies/ml or less in the sample (40particles) with 10,000 copies/ml corresponding to detection of 20particles.

Reaction Platform

In one embodiment, a microfuge tube receives the sample, for example aswab, in a suitable buffer. Alternatively, a reaction platform such as a96 well dish, 384 well dish or other multi-well dish may be used formultiple sample analysis. Alternatively, the sample may be spotted ontoa paper, plastic, or glass surface. The sample may also be introducedinto a microfluidic device, such as a lab-on-a-chip. In this context, anEcho® Liquid Handler (Labcyte Inc., San Jose, Calif.) may be used tohandle fluid samples. Because the one tube reaction is simple, anyautomated liquid handler of device may be used for analysis of multiplesamples. Because the endpoint is a color change, a computerized analysisof a photographic image of the sampling platform or insertion of thereaction platform into a light reader connected to a computer is enabledfor digitizing the reaction platform itself or image thereof. Furtherdetails are described in FIG. 26-FIG. 28F and Example 12.

Preparation of a Master Mix for Use in a LAMP Assay on or in a ReactionPlatform or Reaction Vessel

A master mix can include or be combined with oligonucleotide probes orprimers. A master mix can be added directly to the sample, oralternatively, a portion of the sample can be added to the master mix.The primers and/or probes can be added to the master mix prior to addingthe mixture to the sample or the primers and/or probes can be added tothe sample prior to or after addition of the master mix. Wherelarge-scale analysis of multiple samples is desired, the LAMP primersand/or probes may be incorporated into the LAMP master mix so that allthat is required is to add an aliquot of sample (purified nucleic acidor lysed cell or fluid sample) to the master mix and to raise thetemperature to 60° C.-65° C. for a 15 minute, 30 minute, 45 minute or 60minute incubation time for amplification to occur, to detect a change incolor or fluorescence that defines the presence of the target nucleicacid.

The master mix will contain a DNA polymerase dNTPs and a suitable bufferor water. The master mix may additionally include one or more of thefollowing: probes or primers, a plurality of sets of primers, reversetranscriptase, dUTP and UDG, a helicase, a single strand bindingprotein, a carboxamide, an RNase inhibitor (e.g., murine RNase inhibitoror an aptamer), a pH-sensitive dye or a metallochromic dye such as PAR,and/or manganese ions, and/or guanidine salts. The master mix may havebeen previously stored at −20° C., prepared in a liquid formulation thatis stable at room temperature or lyophilized.

Prevention of Carryover of Contaminating Nucleic Acid Between Samples

Even a very small amount of carryover of previously amplified nucleicacid from a positive sample into a tube that might be negative for thetarget nucleic acid would be very undesirable. For this reason,including dUTP in the dNTPs in a primary amplification reaction resultsin incorporation of UMP into the amplified nucleic acid. If thispreviously amplified DNA then strays into a subsequent sample tube, UDGthat is present will cleave the incoming contaminant DNA. Theestablished method has been to include dUTP with dATP, dCTP, dTTP anddGTP so that a fraction of dT is replaced by a dU in the amplified DNAof the first sample. The second sample is then exposed to UDG prior toamplification and this enzyme creates an abasic site at the incorporateduracil. Consequently, it is not possible to amplify sample 1 amplifiedDNA in sample 2 which would result in a false positive. However, beforesample 2 is amplified, the UDG is temperature inactivated or it wouldadversely affect the desired amplification. It is desirable that thediagnostic test described herein is simple to perform, and also rapidand sensitive. Hence a thermolabile UDG is incorporated into the mastermix along with dUTP so that once the temperature is raised to thetemperature required for amplification, namely 55° C.-75° C., the UDG isinactivated.

A preferred feature of embodiments of the diagnostic test describedherein is inhibition of carryover contamination. In order to confirmthat pH-dependent colorimetric LAMP is not adversely affected by dUTPand thermolabile UDG, a 2×LAMP master mix containing a bufferconcentration of less than 8 mM TRIS buffer and the pH sensitive dye wastested with and without dUTP and Antarctic UDG. It was found that mastermixes containing dUTP and Antarctic UDG yielded substantially the samespeed, sensitivity and specificity observed in the absence of UDG (seeExamples 3 and 4 and FIG. 6A-FIG. 6B). Because prevention of carryoveris important for high throughput screening, preferably, UDG and dUTPshould be included in master mixes for pH colorimetric LAMP detection ofpathogens.

Degradation or Inhibition of Unwanted Proteins in Samples ContainingNucleic Acids in the Absence of the Step of Purifying the Nucleic AcidPrior to LAMP

Thermolabile Proteinase K (see for example, U.S. patent application Ser.No. 16/719,097) and/or a proteinase inhibitor such as murine proteinaseK inhibitor and/or an RNase inhibitor (see for example U.S. provisionalapplication Ser. No. 62/992,921) and/or a DNase inhibitor can be addedto the sample or the reaction tube into which the sample is added. Wherethermolabile Proteinase K is used, it may be inactivated before additionof the master mix. Thermolabile Proteinase K is available from NewEngland Biolabs, Inc, Ipswich, Mass.

Storage of the Master Mix: Dried or Liquid

LAMP master mix containing enzymes, dNTPs, buffer, and pH sensitive dyes(e.g. colorimetric or fluorometric dyes), and/or other dyes that bindmetals (e.g. PAR), may be freeze dried or lyophilized and stored at roomtemperature as a master mix, for example a 2× master mix or 5× mastermix or a 10× master mix until needed. The master mix may then be addedto a solution containing primers and/or sample to be tested for targetnucleic acid in a reaction container, a microfluidic device, a lab on achip device or a matrix such as paper, plastic, or glass.

If the reagents in the master mix are stored in a dried or lyophilizedform, then the pH and buffer concentration of the master mix is notrelevant until the dried master mix is added to the sample or the mastermix is rehydrated. It was here shown that the pH of the buffer ofrehydrated master mix is slightly changed when the master mix isrehydrated so this is taken into account during formulation.

Use of Color or Fluorescence Changes in a Diagnostic Test

pH dependent colorimetric LAMP is shown here to be quick, easy,reliable, and suitable for scale-up in molecular diagnosis of virusesand multicellular parasites and their pathogens. In certain point ofcare formats, it may be desirable to utilize a colorimetric LAMPdiagnostic test in which a color change occurs as a side product ofamplification that does not rely on staining amplified DNA. A desirableformat for measuring a color change is a strip test such as routinelyused for pregnancy tests that rely on antibodies (CVS) or the Quickvue®in-line StrepA test (Quidel Corporation, San Diego, Calif.) orequivalent. Alternatively, a liquid test may be convenient in which thesample is added to a first solution (e.g., a lysis buffer) from which analiquot is removed to a second solution containing the master mix thatis then heated on a small pad provided with the test kit (this could bean equivalent of an activated handwarmer or directions for heating asmall amount of water in a kettle or a heat block). In thesecircumstances a pH change will result in a color change denoting apositive result. Alternatively, in place of a pH dependent colorimetricLAMP assay, a metallochromic dye such as PAR has been found to be usefulin a colorimetric LAMP assay and provides an alternative should bodyfluid to be tested be acidic such as urine. The PAR based assay isdescribed in the figures and examples. Manganese used in the PARcolorimetric endpoint LAMP assay is a suitable ion as it does notnegatively affect the activity of the polymerase or the reversetranscriptase in a LAMP reaction. Because PAR it is not pH sensitive, ithas advantages in a variety of situations in which pH colorimetric LAMPis not best suited as enumerated herein. Either pH colorimetric LAMP orPAR may be used in conjunction with Calcein which also binds tomanganese to give a fluorescent signal and/or with the fluorescent dye,Syto-9. Guanidine salts may also be used in conjunction withcolorimetric LAMP to enhance the sensitivity of the assay.

Master Mix

In embodiments, a master mix may contain a pH sensitive dye, fluorescentdye or PAR, a polymerase, plus optionally a reverse transcriptase forRNA detection (such as for an RNA virus), reversible inhibitors for thepolymerase and reverse transcriptase to reduce unwanted background noiseand increase sensitivity, primers and dNTPs form a reaction mix whencombined with a sample for determining the presence of a target nucleicacid. The master mix is preferably in a concentrated form, for example a2×, 3×, 5×, 7× or 10× master mix where the final amount of master mixafter combination with a sample is 1×.

Some of the considerations in forming the master mix beyond thoseestablished in commercial master mixes (see M1800 from New EnglandBiolabs, Ipswich, Mass.) may include one or more of the followingparameters: (1) the concentration of dye; and (2) the amount of buffer;(3) the pH; (4) dilution of the master mix in the sample; and (5) theproposed incubation time of the reaction mix to which the master mix isadded.

(1) In an example of pH dependent colorimetric LAMP, the concentrationof dye in the master mix should be sufficient such that when dilutedwith sample, it enables unambiguous visual detection of a positivesample. However, the concentration of dye must not be so high as toadversely affect the activity of the polymerase and/or reversetranscriptase. In a preferred embodiment, the visually detectable dye inthe reaction mix (1× master mix plus sample) is in the range of 50μM-200 μM.

(2) The amount of buffer in a liquid concentrated master mix should besufficient to maintain the enzymes and other reagents in a stablecondition but suitable for dilution to detect a color change ifamplification of a target nucleic acid has occurred. It is desirabletherefore not to exceed 5 mM buffer in the reaction mix-samplecombination for pH dependent colorimetric LAMP where bufferconcentrations of greater than 5 mM (e.g., 5 mM Tris) in the reactionmix designed to detect amplification via a pH-based color change werefound to substantially reduce any visually detectable signal foridentifying a positive sample. The buffer concentration was formulatedto meet the 1× amount of master mix to have an equivalence to 0.5 mMTris-5 mM Tris. However, PAR or fluorescence based LAMP do not requirethis limit on buffer concentration.

(3) A pH 8.1-pH 8.5 in the master mix was tested and found to besuitable for phenol red, a pH sensitive dye used in the examples. ThispH could be increased up to a preferred maximum of pH 9 while retaininga visually detectable signal change of pH for a positive sample. The pHcould also be reduced to below pH 8.1 in the master mix to provide achange in color by the dye according to the established pH range ofpink/red phenol red. Lower pH for a detectable color change isundesirable in the event of exposure to atmospheric CO₂ that may acidifythe solution since the master mix is weakly buffered. As the pH isincreased between pH 8.1 and pH 9.0, the color of the negative controlbecomes more pink. Different pH sensitive dyes have different ranges ofpH optima. The pH range is well known in the art (see for example,Tanner et al. (2015) Biotechniques, 58, 59-68).

(4) As described above, LAMP master mixes may be prepared as 2×, 3×, 5×,7× or 10× although 2×, 5× or 10× are the usually preferredconcentrations for easy dilution into a sample. For biological materialcontaining enzyme inhibitors such as blood, sputum, and urine but notnecessarily nasal or buccal swabs, it may be desirable to dilute thesample at least two fold before adding to the appropriately concentratedmaster mix. In addition to this strategy, adjustment of the acidic pH ofurine or the alkali pH of sodium hydroxide treated sputum may bedesirable to ensure that the pH of the sample does not inhibit thedetection of the amplified target nucleic acid in the sample. Buccal andnasal swabs can be placed in water or transport media and added in a 1:1ratio to a volume of master mix resulting in a pH and bufferconcentration as described above. In some circumstances, it may bedesirable to extract the nucleic acid, for example, using Monarch® (NewEngland Biolabs, Ipswich, Mass.), Qiagen or Roche purification kit priorto testing. In these circumstances, the samples can be used in highervolumes especially if eluted from extraction matrices in diluted bufferor water. Lysis buffers are discussed below.

(5) For a rapid test for an infectious agent, it is desirable to analyzea sample directly from a swab or sample matrix containing test nucleicacid without requiring a purification step. Previously, it was shownthat LAMP can detect 2-3 copies of a purified target DNA in a genome inunder 30 minutes and 10 copies under 15 minutes (Tanner, et al. (2015)Biotechniques, 58, 59-68). FIG. 2A-FIG. 2B shows the sensitivity ofassays described herein for nematode parasites as little as 0.01-0.1picograms nematode DNA can be detected. FIG. 3A-FIG. 3B shows detectionof 1.28 femtograms of DNA of a tick-borne pathogen from whole ticks.Embodiments of the test described herein for a target RNA of a virus ina nasal or oral swab can be performed within 15 minutes. Example 1provides an example of pH dependent colorimetric LAMP as used fordetection of viral pathogens such as RNA or DNA viruses for example,SARS-CoV-2 RNA. The results for SARS-CoV-2 RNA in FIG. 21-FIG. 27 showthat the virus can be reliably detected at between 10-40 copies in under60 minutes.

Preparation of Samples for Analysis

Biological material that may contain one or more pathogens can besampled for one or more target nucleic acids in parallel to obtaindistinctive results for each or together for purposes of efficiencyinitially but that might result in secondary tests. For example, where asample is taken from a subject to test for SARS-CoV-2 and influenza, itmight be desirable to perform tests in parallel using one or more primersets that are specific for each target nucleic acid. Samples may beobtained from any biological source. For example, samples may be derivedfrom blood (e.g., from a venous draw), serum, plasma, urine, feces,sputum, hair follicles, lavage, nasal, oral or buccal swabs, and/orsaliva may be used for detecting the pathogen using pH dependent colorLAMP or PAR dependent color LAMP that is not pH dependent. The samplesmay be dried, placed in an aqueous solution that may be selected fromwater or stored in a transport medium, for example saline, TE or atransport medium available from Copan (Murrieta, Calif.). Such productsinclude Sputum Dipper™, SnotBuster™, UriSponge™ and UTM® UniversalTransport System™.

Different types of samples contain variable amounts of RNases that mightbe contained in the biological material. These RNAses are particularconcern if they are released when a diagnostic test requires testing forRNA such as the viral RNA diagnostics for SARS-CoV-2 are performed. TheRNases that are released from the sample can digest the viral RNA fromdisrupted virus of interest reducing the sensitivity of the test.

Detergents are routinely used to disrupt biological material. Examplesof such detergents include Tween and Triton X. These detergents appearto contribute to the observed loss of viral RNA (within 5 minutes atroom temperature) once the biological sample is exposed to thesedetergents. Not wishing to be limited by theory, it is hypothesizedeither that these detergents either activate cell RNases or cause theviruses to break open releasing the viral RNA that is quicklyenzymatically digested by RNAses. One way to prevent RNase digestion isto heat the sample to 95° C. for at least 5 minutes immediately afteradding buffer containing Tween or Triton X to the samples. This heatinactivates the majority of RNAses. However, without some alternativeintervention, the availability of intact sample RNA can be significantlyreduced. Moreover, when handling large numbers of samples, it isproblematic to administer immediate heat treatment (95° C.) withinminutes of adding buffer to avoid loss of RNA. Taking the caps of tubesalone can slow the heat treatment of large numbers of samples. Althoughstorage on ice might help a little, it does not solve the problem of RNAloss.

We have identified a family of surfactants also called an antifoamingagents specifically poloxamers, that can inactivate the virus particlesand either inhibit RNases or do not break open the virus particles, sothat substantially all or a significant portion of the RNA remainsintact for at least 2 hours, 3 hours, 4 hours, 5 hours and up to atleast 6 hours at room temperature after addition of the surfactant priorto a heat step at 95° C. to release viral RNA from virus particles andinactivate the RNase. The observed advantage of the poloxamer PF68 isdescribed in Examples 13-15. It is expected that other related poloxamersurfactants will be similarly effective. Examples of other members ofthis group of surfactants are provided below.

Since the improvement observed relates to the preparation of RNA from asample, it is believed that this group of surfactants can be effectivein the purification of RNA and in improving the sensitivity ofcolorimetric and fluorescent LAMP reactions as well as RT-qPCR reactionsand also in the preparation of samples for nucleic acid sequencing. Theimprovements relate to obtaining RNA from any biological sourceincluding any diagnostic test known in the art for a virus from anybiological material.

The workflow that utilizes these surfactants in a buffer for a sensitivediagnostic assay for an RNA virus is described in FIG. 30 in 3 differentinitial workflows where saliva or other body fluid or buccal or nasalswab is collected in a tube, or in a microwell in a 96 well dish or a384 well dish that may be empty or may contain a buffer with the foamingagent or may contain buffer in a compartment in the tube or well that isreleased on introduction of sample. If the collection tube receives thesample in the absence of buffer, an aliquot can then be transferred toreceiving compartment (tube or a multi-sample microwell dish) where eachwell or tube contains buffer. The tubes or wells containing sample inbuffer containing the poloxamer is then heated to 95° C. for 5 minutesand an aliquot transferred into a fluorescent LAMP, DARQ LAMP,colorimetric LAMP (cLAMP) or RT-qPCR reaction tube to determine thepresence of the virus or viruses. Because of the sensitivity of RNA toRNases, the poloxamer foaming agent may be best suited for adding to thesample at the earliest convenience. It may also be used for DNA virusesor other samples. There was no adverse effect of the carryover of thepoloxamer from sample tubes to the amplification reaction.

Where DARQ (see for example, U.S. Pat. No. 9,074,243) or fluorescent(intercalating dye) LAMP is used to detect target RNA, the poloxameragent is important for improved results while the buffer used along withthe poloxamer does not appear to be of critical importance. For example,a buffer containing TCEP and EDTA may be adequate (for example, 100×buffer containing 0.5 MTCEP (2.5 ml)) 0.5 M EDTA (1 ml) and 1.1 N NaoH(1.5 mls) described in Rabe and Cepko (2020) PNAS vol 117, pp24450-24458) can be used without LiCl. Moreover, while Guanidiniumchloride has proved useful for colorimetric LAMP, it is not required ina fluorescent or DARQ LAMP reaction.

Another factor found to be significant is the volume of buffer in thetubes or wells. The volume in a 96 well dish (e.g., 25 μl) is preferably2× the volume in a 364 well dish (12.5μ).

SARS-CoV-2 LAMP tests were analyzed using a Cq value for real timeanalysis to detect virus and also endpoint +/− analysis. As more isunderstood about SARS-CoV-2, it is important to understand the amount ofvirus in a patient as determined by a nasal or buccal swab or salivathat corresponds to active infection. It has been observed that titersof the order of 2×10⁴ virus particles/ml (20 virus particles/μl) may bedetected at the onset of infection or at the end of a recovery phase ofinfection whereby virus titers in the region of 10⁹-10¹¹ signal aninfectious dose. Therefore, it would be desirable to be able to testindividuals over several days, for example, three times over a singleweek to get a more accurate picture of whether they are spreaders or areabout to be spreaders versus non spreaders. Cost, speed and ease of useof the tests become even more significant in this context. The presentcolorimetric tests (either using pH dependent dyes or fluorescent dyes)described herein meet these criteria where the time to perform a test isless than 1 hour (e.g., 5 minutes to heat a sample to 95° C. and 35-40minutes to perform the LAMP reaction with an immediate endpoint colorreadout). Moreover, the colorimetric LAMP test can readily be performedon multiple samples simultaneously as needed, for example in 96 well or384 well plates with instantaneous detection of positive samples by eyeor by spectrophotometer to detect a color change. Moreover, colorimetricLAMP can consistently detect 100% of samples containing 50-80particles/μl (5-8×10⁴ particles/ml) and is described herein, can detectRNA corresponding to as few as 25 viruses with 90% success rate. Thesedetection ranges of 25-50 virus particles are below the concentrationrequired to detect virus load of an active spreader using saliva, nasalor buccal swabs.

Embodiments are provided for lysis buffers suitable for LAMP. The lysisbuffer was prepared as a 2× mix but could also be prepared as a 4×, 5×or 10× mix or indeed any concentrated form limited only by possibleundesirable precipitation of individual reagents at high concentrations.In one example, the 2× lysis mix described herein comprises 800 mMguanidine HCl, 4% Triton X-100, 80 mM TCEP and 150 mM LiCl at pH 8.0.This 2× lysis mix can be combined with an equal volume of salivaresulting in a final concentration of 400 mM guanidine HCl, 2% TritonX-100, 40 mM TCEP and 75 mM LiCl at pH 8.0. In one embodiment, it wasshown that allowing the saliva-lysis mixture to stand at roomtemperature for 30 minutes increased the sensitivity and reliability ofthe subsequent LAMP reaction. Subsequent to incubation for 30 minutes atroom temperature, the temperature of the mix was raised to 95° C. for 5minutes or 75° C. for 20 minutes or 65° C. for 60 minutes. 2 μl of thesaliva-lysis mix was then added to the standard master mix for LAMPalong with primers.

In a further improvement, it was found that Triton X or Tween could bereplaced with a non-ionic detergent having improved properties withrespect to RNases in saliva. Whereas Triton X and Tween 20 were veryeffective at lysing virus particles at room temperature, thesedetergents appeared to have the effect of exacerbating RNAse activitywith an adverse effect on the sensitivity of an isothermal amplificationperformed on saliva. It was found here that an alkoxylated alcohol whichis an amphiphilic water soluble polymorphic block copolymer ofpoly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), did not havethe undesirable effect on RNAse activity observed for the otherdetergents. A preferable composition would be an ABA triblock copolymer,wherein A is the hydrophilic block PEO and B is the hydrophobic blockPPO. Preferable copolymers have molar mass ratio between the PEO and PPOblocks from 1:9 to 8:2. Examples of such block copolymers are Pluronicdetergents and poloxamers. The class of ABA triblock copolymerscommercially available as Pluronic® (non-proprietary name “poloxamers”)offers a pool of more than 50 amphiphilic, water-soluble and polymorphicmaterials (A=hydrophilic block poly(ethylene oxide) (PEO) andB=hydrophobic block poly(propylene oxide) (PPO). Example 13 describesthe use of Pluronic F68 (CAS [691397-13-4]) (Thermo Fisher, WalthamMass.). The structure is provided in FIG. 38.

Detergents of this class may also be referred asEpoxyethane-epoxypropane copolymer, Ethoxylated propoxylated propyleneglycol, Ethylene glycol-propylene glycol copolymer, Ethyleneglycol-propylene glycol polymer, Ethylene oxide-1,2-propylene oxidecopolymer, Ethylene oxide-propylene oxide copolymer, Ethyleneoxide-propylene oxide copolymer ethylene glycol ether, Ethyleneoxide-propylene oxide copolymer, Ethylene-propylene glycol copolymer,Methyloxirane-oxirane copolymer, Oxirane-methyloxirane copolymer,Oxirane-propylene oxide copolymer, Oxyethylene-oxypropylene copolymer,Oxypropylene-oxyethylene copolymer, Oxypropylene-oxypropylene copolymer,Poly(oxyethylene) poly(oxypropylene) glycol,Poly(oxyethylene)-poly(oxypropylene) polymer,Poly(oxyethylene-oxypropylene) ether, Poly(propylene oxide-ethyleneoxide), Polyethylene oxide-polypropylene oxide, Polyethyleneoxide-polypropylene oxide copolymer, Polyethylene-polypropylene glycol,Polyethylene-propylene glycol diethylene glycol ether,Polyoxyethylenated poly(oxypropylene), Polyoxyethylene oxypropylene,Polyoxyethylene-polyoxypropylene, Polyoxyethylene-polyoxypropylenecopolymer, Polyoxyethylenepropylene glycol ether, Polyoxypropylenepolyoxyethylene propylene glycol ether, Polyoxypropylene-polyoxyethylenecopolymer, Polypropylene glycol-ethylene oxide copolymer, Propyleneglycol-ethylene glycol copolymer, Propylene oxide, polymer with ethyleneoxide, Propylene oxide-ethylene oxide copolymer, Propyleneoxide-ethylene oxide polymer, Propylene oxide-oxirane copolymer, orPropylene oxide-propylene glycol-ethylene oxide copolymer.

PEO-PPO-PEO triblock copolymers are also referred as Etheneoxide-propene oxide triblock copolymer, Ethylene glycol-propylene glycolcopolymer triblock, Ethylene glycol-propylene glycol triblock copolymer,Ethylene oxide-Excenol 1020-propylene oxide triblock copolymer, Ethyleneoxide-propylene oxide triblock copolymer, Ethylene oxide-propyleneoxide-ethylene oxide triblock copolymer, Oxirane-methyloxirane triblockcopolymer, Oxirane-oxypropylene triblock copolymer, Oxirane-propyleneoxide triblock copolymer, Poly(ethylene oxide)-block-poly(propyleneoxide)-block-poly(ethylene oxide), Polyethylene oxide polypropyleneoxide polyethylene oxide triblock copolymer,Polypropylene-polyethylene-polypropylene triblock copolymer, Propyleneglycol-ethylene glycol triblock copolymer, Propylene oxide-ethyleneoxide triblock copolymer, or Propylene oxide-oxirane triblock copolymer.

Most preferable detergents are Pluronic F68 and Poloxamer 188. Otherdetergents of interest are Pluronic L31, Pluronic L61, Pluronic L81,Pluronic L101, Pluronic L121, Pluronic L42, Pluronic L62, Pluronic L72,Pluronic L92, Pluronic L122, Pluronic L43, Pluronic L63, Pluronic L44,Pluronic L64, Pluronic P84, Pluronic P103, Pluronic P123, Pluronic P65,Pluronic P75, Pluronic P85, Pluronic P105, Pluronic F77, Pluronic F87,Pluronic F127, Pluronic F38, Pluronic F88, Pluronic F98, and PluronicF108. Further detergents of interest are Poloxamer 10R5, Poloxamer 1100,Poloxamer 122, Poloxamer 123, Poloxamer 127, Poloxamer 17R4, Poloxamer181, Poloxamer 183, Poloxamer 202, Poloxamer 237, Poloxamer 272,Poloxamer 317, Poloxamer 333, Poloxamer 334, Poloxamer 407, Poloxamer68, Poloxamer F 98, Poloxamer P 188, and Poloxamer P 407.

Further detergents of interest are Acclaim 2220N, Acclaim 4220N, AcclaimPolyol PPO 2220N, Acclaim Polyol PPO 4220N, Aclube 517, Adeka CM 294,Adeka L 61, Adeka Nol 17R2, Adeka Polyether CM 294, Adekanol 25R1,Adekanol F 68, Adekanol L 61, Adekanol L 64, Aduxol VP 11115, Antarox17R4, Antarox 31R1, Antarox L 61, Antarox L 62, Antarox L 64, Antarox SC138, Arlatone F 127G, BASF PE 6800, Basorol 150R1, Basorol PE 6100,Basorol RPE 3110, BL 10500, BL 6400, Blaunon P 0840, Blaunon P 106,Blaunon P 124, Blaunon P 1461, Blaunon P 174, Blaunon P 304, Chemax BP261, Chemex BP 261, CM 294, CRL 1005, Daltocel F 460, EO 106P070E0106,EO 20P070E020, EP 1900, Epan 410, Epan P 45, Epan U 105, EPE 2900, Ethox17R2, Ethox L 122, Ethox L 62, Ethox L 64, EXL 540, EXL 552, EXL 902,ExpertGel 56, ExpertGel EG 56SEC, F 108, F 123, F 126, F 127, F 38, F61, F 68, F 77, F 87, F 88, F 98, H 1000, Hiflex 211, HLB 0.80,Hydropalat WE 3161, Hydropalat WE 3162, Hydropalat WE 3164, HydropalatWE 3966, Kolliphor 188, Kolliphor 407, Kolliphor P 188, Kolliphor P 188Micro, Kolliphor P 237, Kolliphor P 407, Kolliphor P 407 Micro, L 101, L103, L 121, L 123, L 180, L 31, L 35, L 350, L 43, L 44, L 45, L 61, L62, L 62D, L 62LF, L 63, L 64, L 81, LeGoo-endo, Lutrol 127, Lutrol 68,Lutrol F 126, Lutrol F 68, Lutrol F 68NF, Lutrol F 77, Lutrol F 87,Lutrol FC 127, Lutrol L 42,Lutrol L 61, Lutrol L 63, Lutrol L 72, LutrolL 92, Lutrol, Micro 68, Meroxapol 108, Meroxapol 172, Meroxapol 174,Meroxapol 252, Meroxapol 258, Meroxapol 311, Newpol PE 108, Newpol PE61, Newpol PE 75, Newpol PE 78, Nissan Plonon 104, Nissan Plonon 188P,Nissan Plonon 202B, Nissan Plonon 204, Nissan Plonon 208, Nissan Plonon235P, Nissan Plonon 407, Novanik 600/20, Novanik 600/40, Novanik 600/50,P 103, P 104, P 105, P 108, P 123, P 123 surfactant), P 31R1, P 407, P450, P 65, P 68, P 75, P 84, P 85, PE 103, PE 10500, PE 3100, PE 3500,PE 300, PE 61, PE 6100, PE 6120, PE 6800, PE 8100, PE 9400, PEG 100P6,PEP 400-6, Pionin P 1310R, Pionin 2015R, Pionin P 2535, Plonon 104,Plonon 188P, Plonon 202B, Plonon 204, Plonon 208, Plonon 235P, Plonon407, Pluriol PE, Pluriol PE 10100, Pluriol PE 10500, Pluriol PE 1600,Pluriol PE 3100, Pluriol PE 6100, Pluriol PE 6400, Pluriol PE 6810,Pluriol PE 9200, Pluriol PE 9400, Proxanol P 268, PS 137-25, RA 20, RPE1720, RPE 1740, RPE 2520, Slovanik 310, Slovanik S 3040, Slovanik S3070, Surfactant P 123, Surfonic POA 25R2, Synperonic 85, Synperonic F108, Synperonic F 127, Synperonic F 68, Synperonic F 87, Synperonic L64, Synperonic P 85, Synperonic P 94, Synperonic PE-F 103, SynperonicPE-F 108, Synperonic PE-F 127, Synperonic PE-F 88, Synperonic PE-P 85,Synperonic PE/L 121, Synperonic PE/L 61, Synperonic PE/L 64, SynperonicPE/P 65, Tergitol L 61PU, Voranol 222-056N, Voranol 223-060LM, andVoranol EP 1900.

Even further detergents of interest are 333E, 50 MB-26X, 75H380000,75HB1440, 80DE40U, Acclaim 2200N, Actcol ED 36, Actcol ED 56, Actcol MF12, Actcol MF 18, Actcol NF 04, Actinol P 3035, Adeka Carpol MH 150,Adeka Carpol MH 500, Adeka Carpol PH 2000, Adeka L 31, Adeka PolyetherPR 5007, Adeka PR 2008, Adeka PR 3007, Adeka PR 5007, Adekanol L 34,Adekanol L 62, Adekanol NP 1200, Agnique ED 0001, Alcox EP 10, Alcox EP20, Alkan 416, Alkox EP 10, Alkox EP 1010N, Alkox EP 10X, Alkox EP 20,Alkox EP 20X, Antifoam P 21, Atlas SF 131, Balab 615, Berol 370, Berol374, Berol TVM 370, Blaunon EP 1461, Blaunon P 172, Blaunon P 201,Bloatguard, BPE 1500, Breox 50A1000, Breox 50A225, Breox 50A50, Breox75W270, Breox 75W55000, Breox PAG 50A1000, BSP 5000, C 310B, Caradol ED52-03, Carpol 2040, Carpol 2050, CE, CF 0802, Clerol PLB 847, CMC 252,CP 1000, CP 1000 (polyoxyalkylene), CP 1000L, CP 2000, CP 2000 (glycol),CP 2000L, CS-DF 900, D 10, D 21/700, Daltocel F 3001, DDL 400, DE 1, DE1 (demulsifier), DEP 4000E, Desmophen 7100, Desmophen L 2830,Dezemulsionat E 96, Disfoam CC 222, DP 4002E, DP 6000E, DR 4500, ED 36,EL 551, Emkalyx EP 64, Emkalyx L 101, Emkarox VG 1051W, Emkarox VG 217W,Emkarox VG 379W, Emkarox VG 650W, Emulgen PP, Emulgen PP 150, Emulgen PP250, Emulgen PP 290, EP 1660, EP 20, Epan 70, Epan 742, Epan U 102, EpanU 180, EPO 61, EPOB 15F, EPOB 20F, EPOB 30E, EPOB 50E, EPOB 80E, Ethox P104, Excenol 2026T, Excenol 3040, Exocorpol, FT 257, Genapol PF, GenapolPF 10, Genapol PF 20, Genapol PFIO, GPE 2035, Gran Up US 30, HB 126,Hiflex 604, Hiflex D 300, Hiflex DR 4500, Hilube D 550, Hilube TB 1120,IBY 2, Industrol N 3, Jeffol PPG 3706, Jeffox FF 200, Jeffox PPG 2000,K-HN 8200, KE 220, Konion DR 802, Koremul LX 94, KRE 15, KWC-Q, L 5050,Laprol 1502-2-70, Laprol 1601, Laprol 2402C, Laprol 4202-2630, Laprol5002-2630, Laprol D 10, LF 40, LF 40 (polyether), LF 62, LF 62(polyether), Lupranol 2020, Lupranol 2022, Lupranol VP 9243, M 90/20,M-RPE 1293, MAG 540-90DT, Magcyl, Mazu DF 204, MM 8750, Monolan12000E80, Monolan PB, MST 188, Multranol 9111, Multranol 9182, N 480,Nalco PP 10-3340, Nalco SPF-WTB 33, ND 8, Newcol 3280, Newpol PE 34,Nissan Disfoam CC 222, Nissan Nonion A 10R, Nissan Unilube 25DE, NissanUnilube 30DP3B, Nissan Unilube 50DE25, Nissan Unilube 50MB168X, NissanUnilube 50MB26X, Nissan Unilube 50TG32U, Nissan Unilube 60DP5B, NissanUnilube 60MB161, Nissan Unilube 750DE2620, Nissan Unilube 75DE170,Nissan Unilube 75DE5000, Nissan Unilube 80DE120U, Nissan Unilube80DE40E, Nissan Unilube 80DE40U, Nissan Unilube DE 60, Nixolen, NixolenNS 4, Nixolen SL 19, Nixolen SL 2, Nixolen SL 8, Nixolen VS 13, NixolenVS 2600, Nixolen VS 40, Nonion A 10R, Novanik 3010, NSC 63908, Nutek 7C,OHV 112, OHV 168.2, OHV 56.1, OHV 84.1, Oligoether L 1502-2-30, Oxalgon,Oxilube 50/150, Oxilube 50000, PAG, PAG 1, PAG 1 (polyglycol), PAG 2,PEG 600PR, PEG-PPG copolymer, PEP 101, PEPB 2080, PEPB 4555, Pepol AS053X, PEPP 6040, PEPP 8020, PF 10, PF 20, PF 732, PF 80, Pluracare L4370, Pluracol 686, Pluracol V, Pluradyne FL 11, Plurasafe WT 9000H,Pluriol A 2600PE, Pluriol A 4000PE, Pluriol L 64, Pluriol SC 9361,Polosham 188, Poloxalcol, Poloxalene, Poloxalene 2930, Poloxalene L 64,Poloxalkol, Poly-G 55NTP, Poly-G WT 90000, Poly-G WT 9150, Polyglycol D21/150, Polyglycol EP 530, Polyglycol SD 301, Polyglykol D 21/700,Polyglykol PR 600, Polylon 13-5, PPG Diol 3000EO, PPPB 1585, PPPB 3070,Pr 168, PR 3007, PR 5007, PR 600, Prevocell EO, PRO 21, Proksanol,Proxanol, Proxanol 158, Proxanol 168, Proxanol 186, Proxanol 224,Proxanol 228, Proxanol P 168, Proxanol TsL 3, PS 072, PY 1002, RC 102,Regulaid, Rokopol 30P9, Rokopol D 1012E, Rokopol PE 40, Sannix PE 75,SBU 0319, SC 2204, SDT 06E, Separol 29, Separol WF 34, Separol WF 41,Sinoponic P-PE 64, Sipol L 61, SKF 18667, Slovanik, Slovanik 1070/7,Slovanik 610, Slovanik 630, Slovanik 660, Slovanik M, Slovanik M 340,Slovanik PV 670, Slovanik T 310, Slovanik T 320, Slovanik T 630,Supronic B 50, Supronic B 75, Supronic E 400, Supronic E 800, Surflo HS1, Surfonic D 500, Synalox 40D100, Synalox 40D300, Synalox 40D700,Synthionic 80-20, Systol T 154, T 320, Takelac XLR 51, Tdiol 1000, Tdiol2000, Teric PE, Teric PE 40, Teric PE 60, Teric PE 61, Teric PE 62,Teric PE 68, Teric PE 70, TPE 1000, TsL 431, TVM 370, Ucon 25H, Ucon75H, Ucon 75H1400, Ucon 75H450, Ucon 75H90000, Unilube 50DE25, Unilube50MB168X, Unilube 50MB26X, Unilube 60MB161, Unilube 75DE5000, Unilube80DE120U, Unilube 80DE40U, Upol U, Velvetol OE 2NT1, Vepoloxamer,Voranol 222-056, Voranol 5287, Voranol P 2001, Wanefoam RCB 6, WL 1590,Woopol U, WS 661, Wyandotte 7135, X 423, X 427, XD 8379, XUS 15176,Yukol 4813, Z 4, Z 4 (demulsifier), Zeospan 8100L, ZS 2185, ZSN 8100,and ZSP 8100L.

Other amphiphilic block copolymers of interest include combination ofone or more of the selected polymers polyethylene glycol (PEG),poly(D,L-lactide-co-glycolide) (PLGA), poly(lactic acid) (PLA),poly(glutamic acid) (PGA), poly(caprolactone) (PCL),N-(2-hydroxypropyl)-methacrylate (HPMA) copolymers, and poly(aminoacids).

Improved Efficiencies in Workflow Design for Diagnosing Samples fromPatients Infected with a Pathogen

Immobilized reagents can improve a diagnostic workflow. Examples ofimmobilized reagents that may be used for enrichment of a target nucleicacid such as a target RNA contained in a target pathogen or exosome in asample (see description above) may include one or more of: anoligonucleotide for hybridizing to the target nucleic acid, a poly-dToligonucleotide, an oligonucleotide that serves as primer or probe forreverse transcription and/or replication of a target nucleic acid; anucleic acid binding protein such as p19 (see for example U.S. Pat. No.8,753,809), and where the target nucleic acid is DNA, an oligonucleotidereagent that hybridizes top the target DNA, a transcriptional activatorprotein that is sequence specific for the target DNA such as describedin (see for example U.S. Pat. No. 9,963,687), or a nonspecific DNAbinding protein such as a repair protein moiety or a polymerase moiety(such as SSO7); a methyl binding domain for binding methylated DNA; orfor a pathogen containing a target DNA or RNA, a protein specific forbinding a pathogen such as a virion (ACE 2 or GRP78 for SARS-CoV-2 spikeprotein), or an antibody for binding a specific antigen prior lysis ofthe pathogen or exosome so that extraneous material can be removed andthe nucleic acid protected before lysis occurs. Immobilized reagents mayinclude a combination so that the pathogen or exosome may be immobilizedand on release of the nucleic acid from pathogen and exosome possibly inthe same receiving tube, immobilized oligonucleotides can bind targetnucleic acid for further characterization involving amplification togenerate a detectable signal with high sensitivity. Reagents for LAMPmay be immobilized on a substrate instead of the primers or targetnucleic acid. Immobilization of LAMP reagents such as any or all of thestrand displacing polymerase, reverse transcriptase, uracildeglycosylase, nuclease inhibitors and stabilizing reagents may beimmobilized through the use of linkers and/or tags as discussed below.

In one embodiment, a mixture of immobilized reagents may be utilized ina composition where a portion of the immobilized reagents bind to andenrich for one or more pathogens or exosomes or their nucleic acids in asample where the one or more pathogens and/or exosomes may be differenttargets from a single subject (for example, SARS-CoV-2 and Influenzaviruses) or the same or different targets from multiple subjects. In thelatter case, it is desirable to integrate a sample barcode into thereagent oligonucleotide used to capture the target nucleic acids.

Embodiments of the method described herein include washing andreplacement of the biological fluid with a suitable RNAse or DNAse freebuffer containing a lysis reagent, where a second portion of immobilizedreagents captures and enriches the nucleic acid from the lysedparticles.

For example, a nucleic acid from a biological sample includingenvironmental samples can be detected after enrichment in a workflowthat involves a minimum number of steps to produce a sensitive andspecific result. In one example, a reagent is added to an environmentalsample or a body fluid or a solution containing the contents of a swab(see above) to releases nucleic acid from a pathogen or exosomecontained in the sample. The target nucleic acid hybridizes to animmobilized oligonucleotide on a substrate, such as a bead, so thatseparation of the immobilized nucleic acid from the rest of the solutioncan occur and a new solution can be added that has the necessaryreagents for reverse transcription if the nucleic acid is RNA and foramplification, for example, using a polymerase chain reaction (PCR) orby isothermal amplification, for example, LAMP. The amplification eventcan then be detected using fluorescence or color by eye or by means of asimple device. Example 14 describes this approach for a coronavirus. Thehybridized target nucleic acid can be released from the substrate afterenrichment or the immobilized oligonucleotide can act as a primer orprobe so as to function both to enrich the target nucleic acid and toinitiate cDNA synthesis and/or replication.

The use of an immobilized reagent is intended to enhance efficiency ofthe workflow and potentially also enhance sensitivity of an assay fordetecting the pathogen. Others have used immobilization for enrichmentof exons in a genomic DNA sample (see for example U.S. Pat. Nos.9,708,658, 9,567,632, 10,087,481 and 10,246,702). Embodiments describedherein differ from the approaches described in these references becausethey are applicable to detecting a pathogen in a biological fluid andbecause the immobilized reagents may preferably serve as primers orprobes to enrich and reverse transcribe and/or amplify the nucleic acidobtained from the biological sample in a single step.

In present embodiments, immobilized reagents may be provided in alyophilized state and hydrated by the addition of a body fluid sample orsample containing a swab. The rehydrated reagent may include RNaseinhibitors, DNase inhibitors, a detergent and/or surfactant, salts,reducing agents such as DTT or TCEP and proteolytic agents such asProteinase K (such as a heat labile Proteinase K) and salts at asuitable concentration for aiding hybridization that is well understoodin the art and/or destabilizing protein. Examples of a detergent such asa non-ionic detergent includes an amphiphilic water soluble polymorphicblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide)(PPO), Tween 80 or Triton X suitable for disrupting a virus or exosometo release the nucleic acid. The type of detergent and/or surfactant maybe selected depending on what proteolytic agents and reducing agents areincluded to aid in the release of nucleic acid from pathogens orexosomes. If the target nucleic acid is RNA, the use of reducing agentssuch as DTT, TCEP are desirable for stabilizing the RNA. The receivingtube may include in addition to lyophilized reagents or buffercontaining reagents, a concentration of chaotropic salts for denaturingproteins. A heating step may be included where heat can cause denaturingof both proteins and nucleic acids and can be used to (a) inactiveproteins (e.g., DNases and RNases) in the absence of proteinase K and(b) to melt nucleic acid secondary and/or intermolecular hybridizationstructures. However, a heat-denaturing step can be damaging to nucleicacids, particularly in the presence of divalent metals,

Features of the Substrate for Immobilizing Oligonucleotide Reagents

Unless specified otherwise, substrates as used with reference toenrichment refer to immobilization substrates. Substrates may bebiological, non-biological, organic, inorganic or a combination thereof,and may be in the form of solid or porous particles, strands,precipitates, gels, sheets, tubings, spheres, containers, capillaries,pads, slices, films, plates such as microplates, slides, and have anyconvenient shape, including flat, disc, sphere, circle, etc. A substrateas used herein is meant to comprise any material (porous or non-porous,flexible or rigid) material onto which it is desired to capture andimmobilize a reagent oligonucleotide. The substrate may form a colloidalsolution from microbeads. The substrate may be beads that sediment in asolution. The substrate may be magnetic beads that can be concentratedin one part of a tube in response to an external magnet. The substratemay have any desired shape. A spherical shape is convenient because ofthe maximal surface area to volume. The substrate may include anydesirable material known in the art suitable for bindingoligonucleotides including magnetic, paramagnetic or non-magnetic beads.

The substrate for immobilizing reagents may be beads. Examples ofsuitable beads include magnetic or paramagnetic beads are coated withnegatively charged molecules (e.g., carboxyl-containing molecules) whichreversibly bind viral nucleic acids in the presence of a crowding agent(e.g., polyethylene glycol, such as 10-50% PEG) and salt (e.g., NaCl,such as 14 M NaCl). Examples of such beads are SPRI® and AM Pure® beads(Beckman Coulter, Brea, Calif.). Electrostatic reversible binding ofnucleic acids to magnetic or paramagnetic beads may be improved in thepresence of chaotropic salts (e.g., guanidine hydrochloride, guanidiniumthiocyanate, etc.). In some other embodiments, magnetic or paramagneticbeads are coated with positively charged molecules (e.g.,amino-containing molecules) which reversibly bind viral nucleic acids.In some further embodiments, magnetic or paramagnetic beads are coatedwith silica and reversibly binds nucleic acids based on saltconcentration.

Substrate may be modified with secondary molecules to provide surfacesthat facilitate coating of the substrate with a reagent. Examples areprovided below.

The surface of the substrate may be composed of a variety of materials,for example, polymers, plastics, resins, polysaccharides, silica orsilica-based materials, carbon, metals, inorganic glasses, membranes,etc., provided that the surface may support functional groups.Convenient substrates include: glass such as glass slides or glassbeads; plastic such as in microtiter plates; functionalized polymerssuch as polymer beads. The surface of the substrates may be chemicallymodified for example by oxides such as silicon dioxide, tantalumpentoxide or titanium dioxide, or by metals, such as noble metalsurfaces such as gold or silver, copper or aluminum surfaces. Thesubstrate surface may be magnetized for example by Fe, Mn, Ni, Co, andtheir oxides. The substrate may be nanoparticles made of III-Vsemiconductor material (quantum dots) using for example GaN, GaP, GaAs,InP, or InAs) or II-VI semiconductor material using for example ZnO,ZnS, CdS, CdSe, or CdT or Ln-doped fluoride nanocrystals, rareearth-doped oxidic nanomaterials.

The surface of the substrate may inherently contain a linker, crosslinker or spacer, a binder, a functional group, or electrostatic chargeto prepare the substrate for a coating with oligonucleotide reagent. Thesubstrate surface for binding an oligonucleotide can be added oractivated in a coating (such as a polymer coating) of the substrateusing a suitable material. Activation as used herein means amodification of a functional group on the surface of the substrate toenable coupling of a binding agent to the surface. Examples of polymercoatings include any suitable class of compounds, for example,polyethylene glycols, polyethylene imides, polysaccharides,polypeptides, or polynucleotides. Attachment of the polymers to thesubstrate surface may be achieved by a variety of methods which arereadily apparent to a person skilled in the art. For example, polymershaving trichlorosilyl or trisalkoxy groups may be reacted with hydroxylgroups on the substrate surface to form siloxane bonds. Attachment to agold or silver surface may take place via thiol groups on the polymer.Alternatively, the polymer may be attached via an intermediate species,such as a self-assembled monolayer of alkanethiols. The type of polymersselected, and the method selected for attaching the polymers to thesurface, will thus depend on the polymer having suitable reactivity forbeing attached to the substrate surface, and on the properties of thepolymers regarding non-specific adsorption to, especially, DNA and RNA.The functional groups may be present on the polymer or may be added tothe polymer by the addition of single or multiple functional groups.Optionally, a spacer arm can be used to provide flexibility to thebinding oligonucleotide allowing it to interact with its environment ina way which minimizes steric hindrance with the solid support. Thesubstrate can be modified with a binder, e.g., an antibody (or antibodyfragment) or another affinity binder, e.g. streptavidin suitable forbinding an oligonucleotide molecule that has been modified with thecorresponding affinity ligand, e.g. biotin, and another affinity binder,e.g. an antibody recognizing part of the sequence of a biomolecule.

A binder as used herein means any agent that is a member of a specificbinding pair. Examples of binders include agonists and antagonists forcell membranes, toxins and venoms, antigenic determinants, hormones andhormone receptors, steroids, peptides, enzymes, substrates, cofactors,drugs, lectins, sugars, oligonucleotides, oligosaccharides, proteins,glycoproteins, cells, cellular membranes, organelles, cellularreceptors, vitamins, viral epitopes, and immunoglobulins, e.g.,monoclonal and polyclonal antibodies. Examples of binding pairs includebiotin-steptavidin/avidin, hapten/antigen-antibody, carbohydrate-lectin,or others known to those skilled in the art.

Additional examples of specific binding pairs allowing covalent bindingof oligonucleotides to a solid support are e.g. SNAP-tag/AGT andbenzylguanine derivatives (U.S. Pat. Nos. 7,939,284; 8,367,361;7,799,524; 7,888,090; and 8,163,479) or pyrimidine derivatives (U.S.Pat. No. 8,178,314), CLIP-tag/ACT and benzylcytosine derivatives (U.S.Pat. No. 8,227,602), HaloTag and chloroalkane derivatives (Los, et al.Methods Mol Biol., 356:195-208 (2007)), serine-beta-lactamases andbeta-lactam derivatives (International Patent Application PublicationNo. WO 2004/072232). In such as examples, oligonucleotides can befunctionalized with benzylguanine, pyrimidine, benzylcytosine,chloroalkane, or beta-lactam derivatives respectively, and subsequentlybe captured in a solid support modified with SNAP-tag/AGT, CLIP-tag/ACT,HaloTag or serine-beta-lactamases.

Coating of the surface of the substrate with an oligonucleotide reagentby means of a binder may be achieved using an affinity binding pair suchas biotinylated oligonucleotides for binding streptavidin-functionalizedbeads. Other affinity groups on oligonucleotide reagents includedesthiobiotin, avidin, NeutrAvidin, protein A, protein B,maltose-binding protein, chitin, poly-histidine, HA-tag, c-myc tag,FLAG-tag, SNAP-tag, S-tag and glutathione-S-transferase (GST). In oneexample, magnetic or paramagnetic beads are coated with a poly dTsequence (e.g., 10-25 dT oligonucleotide) which is then used as for thedirect binding of poly(A)+ viral RNA. Cap-binding protein attached to animmobilization substrate (an example of such a protein is thetranslation initiation factor 4E, elF4E) can serve as reagents todirectly bind target nucleic acid such as m7G(5′)ppp(5′)N (where N isany canonical nucleotide, including nucleotides with 2′ O-methylation)capped viral RNA (see for example PCT/US2020/031653 for modified caps).

Properties of Oligonucleotides for Use in Immobilizing Target NucleicAcid

The oligonucleotide for hybridizing target nucleic acid should be atleast 6 nucleotides long and may include one or more modifiednucleotides. The oligonucleotide may be designed to function as a probeor primer for reverse transcription and/or amplification. Theoligonucleotide may be any of an RNA, DNA, peptide nucleic acid (PNA), alock nucleic acid (LNA), an unlock nucleic acid (UNA), a triazolenucleic acid, a phosphorothioate oligonucleotide, or a combinationthereof.

Modified nucleotides may improve oligonucleotide chemical stability(e.g., resistance to degradation at basic or acidic pH, at hightemperatures, upon UV radiation, etc.), mechanical stability (e.g.,resistance to degradation following mechanical manipulation such assonication, acoustic shearing, etc.), and/or enzymatic stability (e.g.,resistance to degradation in the presence of a nuclease such as arestriction enzyme, a DNase, a RNase, etc.). Modified nucleotides mayalso improve oligonucleotide ability to hybridize (e.g., through basepairing) with a fully or partially complementary nucleic acid targetedsequence.

Examples of base modifications include 2-aminopurine, 2,6-diaminopurine,5-iodouracil, 5-bromouracil, 5-fluorouracil, 5-hydroxyuracil,5-hydroxymethyluracil, 5-formyluracil, 5-proprynyluracil,5-methylcytosine, 5-hydromethylcytosine, 5-formylcytosine,5-carboxycytosine, 5-iodocytosine, 5-bromocytosine, 5-fluorocytosine,5-proprynylcytosine, 4-ethylcytosine, 5-methylisocytosine,5-hydroxycytosine, 4-methylthymine, thymine glycol, ferrocene thymine,pyrrolo cytosine, inosine, 1-methyl-inosine, 2-methylinosine,5-hydroxybutynl-2′-deoxyuracil (Super T), 8-aza-7-deazaguanine (SuperG), 5-nitroindole, formylindole, isothymine, isoguanine, isocytosine,pseudouracil, 1-methyl-pseudouracil, 5,6-dihydrouracil,5,6-dihydrothymine, 7-methylguanine, 2-methylguanine,2,2-dimethylguanine, 2,2,7-trimethylguanine, 1-methylguanine,hypoxanthine, xanthine, 2-amino-6-(2-thienyl)purine,pyrrole-2-carbaldehyde, 4-thiouracil, 4-thiothymine, 2-thiothymine,5-(3-aminoallyl)-uracil, 5-(carboxy)vinyl-uracil,5-(1-pyrenylethynyl)-uracil, 5-fluoro-4-O-TMP-uracil,5-(C2-EDTA)-uracil, C4-(1,2,4-triazol-1-yl)-uracil, 1-methyladenine,6-methyladenine, 6-thioguanine, thienoguanine, thienouracil,thienocytosine, 7-deaza-guanine or adenine, 8-amino-guanine or adenine,8-oxo-guanine or adenine, 8-bromo-guanine or adenine, ethenoadenine,6-methylguanine, 6-phenylguanine, nebularine, pyrrolidine, andpuromycin. Examples of sugar modifications include but are not limitedto those found in dideoxynucleotides (e.g., ddGTP, ddATP, ddTTP, andddCTP), 2′- or 3′-O-alkyl-nucleotides (e.g., 2′-O-methyl-nucleotides and3′-O-methyl-nucleotides), 2′- or 3′-O-methoxyethyl-nucleotides (MOE),2′- or 3′-fluoro-nucleotides, 2′- or 3′-O-allyl-nucleotides, 2′- or3′-O-propargyl-nucleotides, 2′- or 3′-amine-nucleotides (e.g.,3′-deoxy-3′-amine-nucleotides), 2′- or 3′-O-alkylamine-nucleotides(e.g., 2′-O-ethylamine-nucleotides), 2′- or 3′-O-cyanoethyl-nucleotides,2′- or 3′-O-acetalester-nucleotides,4′-C-aminomethyl-2′-O-methyl-nucleotides, and 2′- or3′-azido-nucleotides (e.g., 3′-deoxy-3′-azide-nucleotides). Furtherexamples of sugar modifications include those found in the monomers thatcomprise the backbone of synthetic nucleic acids such as2′-O,4′-C-methylene-β-D-ribonucleic acids or locked nucleic acids(LNAs), methylene-cLNA, 2′,4′-(N-methoxy)aminomethylene bridged nucleicacids (N-MeO-amino BNA), 2′,4′-aminooxymethylene bridged nucleic acids(N-Me-aminooxy BNA), 2′-O,4′-C-aminomethylene bridged nucleic acids(2′,4′-BNA(NC)), 2′4′-C—(N-methylaminomethylene) bridged nucleic acids(2′,4′-BNA(NC)[NMe]), peptide nucleic acids (PNA), triazole nucleicacids, morpholine nucleic acids, amide-linked nucleic acids,1,5-anhydrohexitol nucleic acids (HNA), cyclohexenyl nucleic acids(CeNA), arabinose nucleic acids (ANA), 2′-fluoro-arabinose nucleic acids(FANA), α-L-threofuranosyl nucleic acids (TNA), 4′-thioribose nucleicacids (4'S-RNA), 2′-fluoro-4′-thioarabinose nucleic acids (4'S-FANA),4′-selenoribose nucleic acids (4′Se-RNA), oxepane nucleic acids (ONA),and methanocarba nucleic acids (MC).

Attachment of Oligonucleotides to the Substrate

Oligonucleotides as described herein may be attached to a substrate viaa linker, a functional group, by cross linking, or by electrostaticcharge that enables coating of the substrate with the reagentoligonucleotide but avoids non-specific binding of the targetoligonucleotide. To immobilize the oligonucleotide on the solid supportsurface, the activated functional groups on the surface may be presenton the predefined regions only, or alternatively on the entire surface,are reacted selectively with the functional groups present in theoligonucleotide molecules. The necessary reaction conditions, includingtime, temperature, pH, solvent(s), additives, etc. will depend on interalia the particular species used and appropriate conditions for eachparticular situation will readily be apparent to the skilled person.

(a) Linker: The oligonucleotide reagent may be attached to the substratethrough the oligonucleotide 5′ end, 3′ end or through an internalnucleotide. The oligonucleotide may be attached to the solid supportthrough a linear or branched linker. The linker separating theoligonucleotide from the substrate may serve as steric spacer and doesnot necessarily have to be of defined length. Examples of suitablelinkers may be selected from any of the hetero-bifunctionalcross-linking molecules described by Hermanson, Bioconjugate Techniques,2nd Ed; Academic Press: London, Bioconjugate Reagents, pp 276-335(2008), incorporated by reference. The linker may be a flexible linkerconnecting the solid support to one or a plurality of same or differentoligonucleotides.

(b) Functional Group or Cross Linking: A variety of methods are knownfor attaching an oligonucleotide to a substrate, including covalentbonding to the support surface and non-covalent interaction (binding byadsorption, e. g. cationic surfaces) of the oligonucleotide with thesurface. Typically, covalent immobilization involves the reaction of anactive functional group on the oligonucleotide with an activatedfunctional group on the solid surface. Examples of reactive functionalgroups include amines, hydroxylamines, hydrazines, hydrazides, thiols,phosphines, isothiocyanates, isocyanates, N-hydroxysuccinimide (NHS)esters, carbodiimides, thioesters, haloacetyl derivatives, sulfonylchlorides, nitro- and dinitrophenyl esters, tosylates, mesylates,triflates, maleimides, disulfides, carboxyl groups, hydroxyl groups,carbonyldiimidazoles, epoxides, aldehydes, acyl-aldehydes, ketones,azides, alkynes, alkenes, nitrones, tetrazines, isonitriles, tetrazoles,and boronates. Examples of such reactions include the reaction betweenan amine and an activated carboxy group forming an amide, between athiol and a maleimide forming a thioether bond, between an azide and analkyne derivative undergoing a 1,3-dipolar cycloaddition reaction,between an amine and an epoxy group, between an amine and another aminefunctional group reacting with an added bifunctional linker reagent ofthe type of activated bis-dicarboxylic acid derivative giving rise totwo amide bonds, or other combinations known in the art. Otherreactions, such as UV-mediated cross-linking can be used for covalentattachment of oligonucleotide to solid supports. Functionalization ofsurfaces with biological materials can also be used for attachingoligonucleotides to solid supports.

Alternatively, oligonucleotides can be specifically or nonspecificallyattached to SNAP-tag/AGT, CLIP-tag/ACT, HaloTag orserine-beta-lactamases and subsequently be captured in a solid supportfunctionalized with benzylguanine, pyrimidine, benzylcytosine,chloroalkane, or beta-lactam derivatives, respectively.

To immobilize the oligonucleotide reagent on the surface of a substrate,the activated surface functional groups may be present in predefinedregions of the substrate only, or alternatively on the entire surface ofthe substrate, and can be reacted selectively with functional groupspresent in the oligonucleotide molecules. The necessary reactionconditions, including time, temperature, pH, solvent(s), additives, etc.will depend on inter alia the particular species used and appropriateconditions for each particular situation will readily be apparent to theskilled person.

Reagent Oligonucleotides

Oligonucleotides can be synthesized to incorporate a desired functionalgroup. Individual nucleotides can be modified either chemically orenzymatically with any type of functional group in order to provide thedesired reactivity. This chemical or enzymatic functionalization can beextended to DNA and RNA molecules.

For example, oligonucleotides can be specifically or nonspecificallyattached to SNAP-tag/AGT, CLIP-tag/ACT, HaloTag orserine-beta-lactamases and subsequently be captured on a substratefunctionalized with benzylguanine, pyrimidine, benzylcytosine,chloroalkane, or beta-lactam derivatives, respectively. Further examplesof specific binding pairs allowing covalent binding of oligonucleotidesto a solid support are acyl carrier proteins and modifications thereof(binder proteins), which are coupled to a phosphopantheteine subunitfrom Coenzyme A (binder substrate) by a synthase protein (see forexample, U.S. Pat. No. 7,666,612).

Examples of proteins or fragments thereof allowing convenient binding ofDNA to a solid support are e.g. chitin binding domain (CBD) (see forexample, US 584,247 and U.S. Pat. No. 7,732,565 and New England Biolabs)maltose binding protein (MBP) (See for example U.S. Pat. Nos. 5,643,758and 8,623,615 and NEBExpress® MBP Fusion system (New England Biolabs,Ipswich, Mass.) omitting TEV cleavage), glycoproteins,transglutaminases, dihydrofolate reductases, glutathione-S-transferaseal (GST), FLAG tags, S-tags, His-tags, and others known to those skilledin the art. Typically, an oligonucleotide is modified with a moleculewhich is one part of a specific binding pair and capable of specificallybinding to a partner covalently or noncovalently attached to a solidsupport.

Coating of magnetic or paramagnetic beads with oligonucleotide primersmay be achieved using an affinity binding pair, such as instreptavidin-functionalized beads and biotinylated oligonucleotides.Other preferred affinity groups include desthiobiotin, avidin,NeutrAvidin, protein A, protein B, maltose-binding protein, chitin,poly-histidine, HA-tag, c-myc tag, FLAG-tag, SNAP-tag, S-tag andglutathione-S-transferase (GST). In some other preferred embodiments,magnetic or paramagnetic beads are coated with a poly dT sequence (e.g.10-25 dT oligonucleotide) which can then be used as for the directbinding of a target nucleic acid having a poly A tail such as a poly(A)+viral RNA. Further preferred embodiments include magnetic orparamagnetic beads coated with a cap-binding protein (an example of sucha protein is the translation initiation factor 4E, elF4E) which can beused for the direct binding of m7G(5′)ppp(5′)N (where N is any canonicalnucleotide, including nucleotides with 2′ O-methylation) capped viralnucleic acid (see for example PCT/US2020/031653).

Reactors for Analyzing Saliva.

A further consideration is the use of immobilized enzymes to improve acoronavirus testing workflow that utilizes saliva (or other bodilyfluid) as a starting material to test for the presence of virus and usesLAMP to amplify nucleic acids derived from any SARS-CoV-2 virus presentin the saliva to determine whether an individual is infected. In suchembodiments, the enzymes involved in reverse transcription and LAMP arestably and efficiently immobilized on a microchip or any column reactoror fluid channel network (including low-cost polymers, such aselastomer, and paper). The enzymes may be irreversibly adsorbed orcovalently linked to the solid surface using any one of the methodsdescribed in this invention. The saliva sample and any other buffers andreagents are flowed through the microreactor by a peristaltic pump. Anyunwanted biological material may be separated from the desired viralgenome by means of an integrated in-line microchannel containing anappropriate separation material. The separation material may beirreversibly adsorbed or covalently linked to channel to permitseparation through one or more of the following techniques:size-selection, ion exchange, size-exclusion, affinity selection,hybridization, and liquid chromatography. The saliva nucleic acid isthen flowed through one or more channel(s) where the enzymes involved inreverse transcription and LAMP are immobilized. A readout of the LAMPreaction will indicate or not the presence of viral genome.

Analysis of Assay Endpoints for Individual or Multiple Samples

Individual samples were tested herein in strip well tubes or 96 wellplates with positive and negative outcomes observed by eye, and alsowith a spectrophotometer (SpectraMax) using dual wavelengths thatcaptured signal from 560 nm (red) to 432 nm (yellow) from each 384 wellplate. The data was then presented on a computer readout. The resultsparticularly with a cooling step between the LAMP reaction and thereading of plates resulted in increased sensitivity in detecting as fewas 20 copies of viral RNA/well in 100% of samples tested.

An example of an automated workflow from sample to collection to outputwas envisaged in FIG. 28A-FIG. 28F. Although there are manypossibilities in the uses of robotic devices for individual steps, theexemplified workflow is predicted to have a capability to perform100,000 reactions in 20 hours.

There is some flexibility based on the parameters described herein thatwill be apparent to a person of ordinary skill in the art as to one ormore modifications selected from: the source of a sample from a patient;sample storage; sample lysis containing for example, guanidine salts,reducing agent and optionally detergent to provide RNA or DNA that maythen be directly amplified or sequenced; the type of sequencing platformselected as to whether it is a single molecule sequencer such as OxfordNanopore or a sequencer of libraries with adapters such as required byan Illumina sequencing platform; the type of amplification reactionselected according to a high through put time and cost efficient LAMPbased reaction using appropriately selected single or multiple primersets or an RT-qPCR real time slightly more sensitive but less time andcost efficient than LAMP; carry over prevention; the optimization of theamplification reactions with respect to pH, buffer content includingguanidine salts, and concentration, of reagents; end point color changebe it pH dependent or dependent on a chemical reaction or fluorescence;and a suitable reader for distinguishing positives from negative samplesin a binary determination and a rapid read out.

LAMP sensitivity has been improved by reducing background and enhancingsignal and these improvements can be followed through to the presentassay. See for example: U.S. Pat. Nos. 9,121,046, 9,546,358, 9,074,249,9,074,243, 9,157,073, and 9,127,258 in addition to U.S. Pat. Nos.9,580,748, 9,034,606, and 10,597,647 all incorporated in entirety byreference. These modifications can be incorporated into the colorimetricLAMP assay described herein to improve the detection of pathogens evenfurther. U.S. Pat. No. 10,253,357 is also incorporated by reference.

The diagnostic test of whether a pathogen is present in a sample can bescaled up without any difficulty so that any of 1-1000s of reactions canbe performed at the same time. If the reactions are performed in 96 welldishes or in 1000 well dishes, a robot liquid handler can add master mixto each well and then the swab sample and a computer system can recordthe color changes and the location of the well testing positive. Forindividual or small numbers of samples, the reactions might be performedin microfuge or PCR tubes. The entire diagnostic test can be completedwithin 4 hours, for example in less than 3 hours, for example less than2 hours, for example less than 1 hour from the time of taking the sampleto obtaining a result. The diagnostic test can be performed in adoctor's office or even at home.

In certain embodiments the master mix including enzymes, dyes, primersand nucleotides may be dried onto a solid matrix such as paper so thataddition of a measured droplet of the sample onto the paper and aheating step even on a surface heated by a homemade water bath, or smallheating block that can be transported in a backpack for environmentaluse results in a color change to indicate a result. Alternatively, themaster mix may be freeze dried or lyophilized and contained in a tubeready for addition of a sample in the home or clinic.

Embodiments include incorporating amplification reagents in a facemaskpossibly immobilized on beads such that when droplets of salivacontaining virus contact the beads, fluorescence results from anisothermal amplification reaction. Whereas LAMP as described inembodiments herein requires a 65° C. temperature step, this requirementmay be circumvented in the future for LAMP or by use of other isothermalamplification methods. Alternatively, the combination of a salivadroplet containing a virus particle interacting with immobilized regenton a bead might trigger an exothermic chemical reaction. The higher thevirus load, the stronger the signal that would result fromamplification. Alternatively, the signal from the production of hydrogenions or change in flow of electrons (that result from amplification aspyrophosphates are released when dNTPs are incorporated duringamplification reaction) that generates a visual signal might in turntrigger a sound wave that is amplified resulting in an audible sound.Such microelectronic technology already exists in a different formatsand could be constructed using synthetic biology constructed circuits.An audible sound could alert the wearer of the mask of 3^(rd) partyreleased virus without the need to remove the mask while a visual signalwould alert others of the wearer of the mask being infected.

In one embodiment, a discrete portion of a face mask may contain achamber containing lysis reagent in a dried or liquid form so thatincoming saliva droplets will be lysed and the polynucleotides released.

Embodiments describing improvements in the LAMP reaction that includecarryover prevention, RNase inhibition, enhancement of sensitivity andrate of the LAMP reaction by the use of guanidine salts and/or reducedNaCl or KCl in the buffer, selecting primer sets and multiplexing primersets, lyophilization of reagents can be combined in any combination forpurposes of automation of large numbers of assays for genomic studies,gene expression studies or epidemiology analysis as well as point ofcare and miniaturization of tests to act as environmental sensors ofpathogens.

Multiplexing as discussed herein can be achieved by pooling patientsamples or purified nucleic acids from different sources and testing forthe presence of a single species of target sequence such as contained ina single infectious agent such as SARS-CoV-2. The samples in the poolcan be differentiated by the use of random sequence identifiers referredto in the art as “unique identifiers” (UIDs), “unique molecularidentifiers” (UMIs) or “degenerate base regions” (DBRs) etc. pH orfluorescent colorimetric LAMP as discussed herein is well suited foranalyzing pooled samples.

Multiplexing may also be used on a single vertebrate subject sample, todetermine the presence any one of several different target nucleicagents such as genes, or infectious agents such as viruses, bacterial orfungi such as respiratory viruses such a coronavirus, a rhinovirus, arespiratory syncytial virus, an Influenza virus, a parainfluenza virus,a metapneumovirus, an adenovirus and a bocavirus. One example of amultiplex test would include Influenza A and B and SARS-CoV-2 that mayoccur together or separately in humans in winter seasons in northern andtemperate zones. Other vertebrates may include mammals such as wild ordomestic mammals such as bats, pigs or birds that are carriers ofviruses that are pathogenic for humans.

Multiplex LAMP for multiple targets is preferably performed usingfluorescent colorimetric LAMP for example using “Detection ofAmplification by Releasing of Quenching” (DARQ) that supplements astandard LAMP primer set with a pair of oligonucleotides in duplex formfor detection (see for example, U.S. Pat. No. 9,074,243). The DARQoligonucleotide duplex consists of a 5′-modified version of the FIPprimer (Q-FIP) annealed to a 3′-modified oligonucleotide represent theF1 region (Fd), complementary to the FIP in its 5′ section. Thefluorescent dye and quencher can be present either on the 5′ orcomplementary 3′ oligonucleotide of the duplex. The two modificationsare selected to be dark quencher-fluorophore pairs, and when thequenched FIP is incorporated into the LAMP product, subsequentamplification displaces the Fd oligo, releasing quenching and producingfluorescent signal specific to the label and target of interest. A listof some fluorescent dyes and quenchers are provided in U.S. Pat. No.9,074,243.

Other versions of this approach for single targets have been used,moving the quencher and fluorophore to a Loop primer or incorporatedinto the amplification products and detected after the reaction (see forexample, Tanner, et al. Biotechniques 53(2), 81-89 (2012), Curtis, etal. J. Virol Methods 255 91-97 (2018) and Yaren, et al. medRxivdoi:10.1101/2020.09.29. 20204131 2020.2009.2029.20204131 (2020)).

As the SARS-CoV-2 pandemic continues into flu season, the ability todistinguish which causative agent is responsible for what can manifestas very similar symptoms will be of great importance for diagnostics anddisease surveillance. A patient presenting with respiratory symptoms mayhave SARS-CoV-2, Influenza A or B, RSV, a rhinovirus, etc. and using onetest to identify multiple infectious agents in the same procedure willsave time and cost. Most importantly, it gives a more definitivediagnostic identification. Example 17 describe a diagnostic assay inwhich multiple LAMP primer sets were used in a single tube and togetherwere capable of detecting the two most common influenza strains andSARS-CoV-2 (SARS-CoV-2, Influenza A, Influenza B, and an internalcontrol). The diagnostic assay also conformed to the useful propertiesof speed, sensitivity and non-interference thereby expanding the utilityof the widely-used LAMP chemistry to a multiplex diagnostic setting (seefor example, FIGS. 31A-31E and 32A-32C).

The selection of appropriate targets, confirmation of minimuminterference between primer sets, and choice of a suitable internalcontrol to verify LAMP was achieved to enable a successful multiplexreaction to be performed. It was found that an internal control (acellular RNA such as actin (ACTB) could be effectively incorporated intoa multiplex DARQ LAMP reaction without interference providing that theconcentration of the primer set for detecting the control was lower thanthe concentration used to detect respiratory pathogens in the sample. Itwas found that the concentration of the control primer set shouldpreferably be between 25% and 80% of the concentration of the primersets for pathogen targets. For example, the preferred range was 50%-80%or 50%-75%.

Criteria were identified to establish the number and type of fluorescentdyes including wavelength emission spectrum, sensitivity of emittedsignal from the unquenched sample and suitable multichannel devices forreading endpoint or continuous signals from multiple differentfluorescent labels from a single reaction vessel.

It was confirmed that multiplex DARQ LAMP using primer sets for multiplevirus targets and an internal control had advantageous propertiesincluding:

-   -   (a) The rate of the DARQ LAMP showed a target dosage response        that was approximately equivalent to the conventional LAMP        monitored by the intercalating dye-SYTO-9. SARS-CoV-2 RNA was        used at various concentrations with the E1 primer set and ACTB        (actin) control primer set.    -   (b) The detection sensitivity of multiplex DARQ LAMP was        determined to be similar to standard single target LAMP and DARQ        LAMP under the same conditions as in (a). The addition of a        second primer set did not significantly alter the detection        sensitivity.    -   (c) End point fluorescence measurements could be reliably used        to distinguish between positive and negative samples using a        threshold value that was set based on negative (background)        values. Positives were matched to those called during real time        monitoring. The end point result demonstrates compatibility of        endpoint, plate-reader measurements with multiplex DARQ LAMP,        enabling use on a wider range of instrument types and increasing        potential test throughput compared with real time detection. A        limitation of real time detection is that it relies on certain        detection instruments that are tied up during the entire        incubation time of samples thereby negatively impacting sample        throughput.    -   (d) DARQ LAMP could be used for detecting 5 different targets        using 5 different sets of LAMP.        -   primers. The limitation of 5 was set by the number of            channels in a BioTek reader (BioTek, Winooski, Vt.). BioTek            Synergy Neo2 microplate reader was used detecting a            fluorescence signal for 5-FAM (Excitation, 484/20, Emission            530/25, Signal Gain 75), HEX (524/20, 565/20, 75), 5-ROX            (569/20, 615/25, 85) and Cy5 (640/20, 682/20, 75). Indeed,            there are as many as a hundred different fluorescent dyes            that have different wavelengths across the 480 nm-640 nm            spectrum. Providing there is at least 20 nm-40 nm of            wavelength between individual fluorescence peaks, a reader            might accommodate primer sets and targets that exceed 5            targets in a single multiplex reaction. Primer sets against            two different Influenza RNA targets and one SARS-CoV-2 RNA            target and a single internal control were used to establish            the feasibility of multiplexing.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Still, certain elements may bedefined for the sake of clarity and ease of reference. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present teachings will be limited onlyby the appended claims.

Sources of commonly understood terms and symbols may include: standardtreatises and texts such as Kornberg and Baker, DNA Replication, SecondEdition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, SecondEdition (Worth Publishers, New York, 1975); Strachan and Read, HumanMolecular Genetics, Second Edition (Wiley-Liss, New York, 1999);Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach(Oxford University Press, New York, 1991); Gait, editor, OligonucleotideSynthesis: A Practical Approach (IRL Press, Oxford, 1984); Singleton, etal., Dictionary of Microbiology and Molecular Biology, 2nd ed., JohnWiley and Sons, New York (1994), and Hale & Markham, the Harper CollinsDictionary of Biology, Harper Perennial, N.Y. (1991) and the like.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. For example, the term “a protein” refers to one or moreprotein, i.e., a single protein and multiple proteins. It is furthernoted that the claims can be drafted to exclude any optional element.

Aspects of the present disclosure can be further understood in light ofthe embodiments, section headings, figures, descriptions, and examples,none of which should be construed as limiting the scope of the presentdisclosure in any way. Accordingly, the claims set forth below should beconstrued in view of the full breadth and spirit of the disclosure.

Each of the individual embodiments described and illustrated herein hasdiscrete components and features which can be readily separated from orcombined with the features of any of the other several embodimentswithout departing from the scope or spirit of the present teachings. Anyrecited method can be carried out in the order of events recited or inany other order which is logically possible.

Numeric ranges are inclusive of the numbers defining the range. Allnumbers should be understood to encompass the midpoint of the integerabove and below the integer i.e. the number 2 encompasses 1.5-2.5. Thenumber 2.5 encompasses 2.45-2.55 etc. When sample numerical values areprovided, they may represent, unless specified otherwise, anintermediate value in a range of values. If specified, an individualnumerical value may represent an extreme point in a range. Where aplurality of numerical values are provided these may represent theextremes of a range unless specified. If specified, these values mayrepresent intermediate values in a range.

The term “non-naturally occurring” as used herein refers to acomposition that does not exist in nature. A “non-naturally occurring”protein may have an amino acid sequence that is different from anaturally occurring amino acid sequence for example, one or more aminoacid substitutions, deletions or insertions at the N-terminus, theC-terminus and/or between the N- and C-termini of the protein. Hence thenon-naturally occurring protein may have less than 100% sequenceidentity to the amino acid sequence of a naturally occurring proteinalthough it may have at least 80%, at least 85%, at least 90%, at least95%, at least 97%, at least 98%, at least 98.5% or at least 99% identityto the naturally occurring amino acid sequence. In certain cases, anon-naturally occurring protein may include a protein that has apost-translational modification pattern that is different from theprotein in its natural state for example, an N-terminal methionine ormay lack one or more post-translational modifications (e.g.,glycosylation, 5 phosphorylation, etc.) if it is produced by a different(e.g., bacterial) cell.

In the context of a nucleic acid, the term “non-naturally occurring”refers to a nucleic acid that contains: a) a sequence of nucleotidesthat is different from a nucleic acid in its natural state (i.e., havingless than 100% sequence identity to a naturally occurring nucleic acidsequence); b) one or more non-naturally occurring nucleotide monomers(which may result in a non-natural backbone or sugar that is not G, A, Tor C); and/or c) may contain one or more other modifications (e.g., anadded label or other moiety) to the 5′-end, the 3′ end, and/or betweenthe 5′- and 3′-ends of the nucleic acid.

In the context of a composition, the term “non-naturally occurring”refers to: (a) a combination of components that are not combined bynature, e.g., because they are at different locations, in differentcells or different cell compartments; (b) a combination of componentsthat have relative concentrations that are not found in nature; (c) acombination that lacks something that is usually associated with one ofthe components in nature; (d) a combination that is in a form that isnot found in nature, e.g., dried, freeze dried, crystalline, aqueous;and/or (e) a combination that contains a component that is not found innature. For example, a composition may contain a “non-naturallyoccurring” buffering agent (e.g., Tris, HEPES, TAPS, MOPS, tricine orMES), a detergent, a dye, a reaction enhancer or inhibitor, an oxidizingagent, a reducing agent, a solvent or a preservative that is not foundin nature. The non-naturally occurring polymerase may be purified sothat it does not contain DNases, RNases or other proteins withundesirable enzyme activity or undesirable small molecules that couldadversely affect the sample substrate or reaction kinetics.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by referenceincluding: U.S. Provisional Ser. Nos. 62/988,696, 63/001,909,63/013,422, 63/022,303, 63/027,216, 63/048,556, 63/059,891, 63/068,564,63/106,120 and 63/165,465 and U.S. Ser. Nos. 16/938,575, 17/122,979,17/178,395 and 17/221,541.

Certain embodiments of the invention are summarized below:

-   -   1. A master mix comprising: a strand displacing polymerase        suitable for Loop-Mediated Isothermal Amplification (LAMP) of        DNA; dATP, dGTP, dCTP, and dTTP; at least one reagent that        changes color or provides fluorescence if amplification occurs;        wherein the master mix is either dried or in a weakly buffered        solution at a starting pH which is measurably altered during        amplification.    -   2. The master mix according to 1, immediately above, further        comprising a reverse transcriptase.    -   3. The master mix according to 2, immediately above, wherein the        reverse transcriptase in selected from an HIV derived reverse        transcriptase, an intron expressed reverse transcriptase and a        reverse transcriptase variant of mouse murine virus.    -   4. The master mix according to any of 1 through 3 above, wherein        the starting pH is in the range of pH 7.5-pH 9.0.    -   5. The master mix according to 4, wherein the starting pH is        preferably in the range of pH 7.8-pH 8.5.    -   6. The master mix according to any of 1 through 5, wherein the        master mix is in a weakly buffered solution of 5 mM Tris or        less.    -   7. The master mix according to any of 1 through 6, wherein the        master mix further comprises an aptamer for regulating the        activity of the strand displacing polymerase.    -   8. The master mix according to any of 1 through 7, wherein the        master mix further comprises one or more RNase inhibitors.    -   9. The master mix according to 8, wherein the RNase inhibitors        comprise an aptamer for inhibiting RNase A, an aptamer for        inhibiting RNase I and/or a protein inhibitor of RNAse.    -   10. The master mix according to 1 through 9, further comprising        at least one set of primers having specificity for a target        nucleic acid.    -   11. The master mix according to 1 through 10, further comprising        a plurality of sets of primers having specificity for a target        nucleic acid.    -   12. The master mix according to 1 through 11, wherein the at        least one reagent includes a dye that is pH sensitive and        changes color after an amplification reaction in which the pH is        altered.    -   13. The master mix according to 1 through 12 further comprising        dUTP.    -   14. The master mix according to 1 through 13, wherein the at        least one reagent includes a dye that is a metallochromic        indicator.    -   15. The master mix according to 1 through 14, wherein the at        least one reagent comprises a metallochromic dye and a        fluorescent dye.    -   16. The master mix according to 1 through 15, wherein the        metallochromic indicator is 4-(2-pyridylazo) resorcinol (PAR).    -   17. The master mix according to 1 through 16, wherein the master        mix is freeze dried, air dried, or lyophilized.    -   18. The master mix according to 1 through 17, wherein the master        mix is immobilized; such as wherein the master mix is        immobilized on paper, or on a natural or synthetic polymer.    -   19. The master mix according to any of 1 through 16, wherein the        master mix is in liquid form in a reaction container.    -   20. The master mix according to any of 1 through 19, further        comprising dUTP.    -   21. A method for determining whether a target nucleic acid is        present in a sample, comprising:        -   (a) placing the sample into an aqueous solution in a            container;        -   (b) bringing an aliquot of the sample into contact with a            master mix according to any of claims 1 through 20 to form a            reaction mixture; and        -   (c) determining whether the target nucleic acid is present            in the sample by a change in the color or fluorescence of            the mixture.    -   22. The method according to 21, wherein the sample is a clinical        sample, such as a sample of a body fluid or a sample taken from        a swab, an environmental sample, or a sample of purified nucleic        acid.    -   23. The method of 21 or 22, wherein the target nucleic acid is:        associated with a pathogen or is a diagnostic target for        pathogenesis; associated with gene expression; or an indicator        of a metabolic response to a pharmaceutical preparation or        allergen.    -   24. The method of 23, wherein the target nucleic acid is an RNA.    -   25. The method according to 23 or 24, wherein the target nucleic        acid is associated with a pathogen.    -   26. The method according to 25, wherein the pathogen is a virus.    -   27. The method according to 26, wherein the virus is an RNA        virus.    -   28. The method of 21 or 22, wherein the target nucleic acid is        DNA, and the method is for determining genetic loci correlated        to a phenotype.    -   29. The method of 28, wherein the genetic loci are selected from        the group consisting of a single nucleotide polymorphism (SNP)        in a genome, an exon, or gene in the genome.    -   30. The method according to any of 21 through 29, wherein the        container contains multiple compartments each for analyzing a        separate sample.    -   31. The method according to any of 21 through 30, wherein the        change in color or fluorescence of the mixture can be digitized        and recorded by a computer.    -   32. A composition, comprising: one or more primer sets suitable        for amplification, the primer sets having specificity for a        single template nucleic acid of interest; and a buffer        containing a molecule comprising C—(NH₂)₂NH⁺.    -   33. The composition according to 32, wherein the molecule        comprising C—(NH₂)₂NH⁺ is selected from guanidine hydrochloride,        guanidine thiocyanate, arginine, and guanidine sulfate.    -   34. The composition according to 32 or 33, wherein the one or        more primer sets are primer sets for Loop-Mediated Isothermal        Amplification (LAMP).    -   35. The composition according to any of 32 through 34, wherein        the one or more primer sets are two or three or four primer sets        having specificity for a single template nucleic acid.    -   36. The composition according to any of 32 through 36, further        comprising one or more reagents selected from an RNAse        inhibitor, a reverse transcriptase, a thermolabile Proteinase K,        and a polymerase.    -   37. The composition according to any of 32 through 37, further        comprising dNTPs; and optionally further comprising a reporter        molecule for detecting amplification in the presence of a target        nucleic acid.    -   38. The composition according to 37, wherein the reporter        molecule comprises a metallochromic dye.    -   39. The composition according to 38, wherein the metallochromic        indicator is 4-(2-pyridylazo) resorcinol (PAR).    -   40. A method of isothermal amplification (e.g., LAMP),        comprising: (a) adding the composition according to any of 1        through 20, and 32 through 38, to a sample comprising a target        nucleic acid; and (b) detecting whether the target nucleic acid        is present in the sample.    -   41. A method for detecting a target nucleic acid in a biological        sample, comprising:        -   (a) treating the biological sample with a lysis mixture            comprising a C—(NH₂)₂NH⁺ salt, and a reducing agent; at an            elevated temperature for an effective period of time;        -   (b) adding an aliquot of the sample from (a) into a master            mix according to any of 1 through 20;        -   (c) incubating the mixture under conditions for Loop            mediated amplification (LAMP) to permit a color change in            the presence of a detectable amount of a target nucleic            acid; and        -   (d) determining whether the sample contains the target            nucleic acid.    -   42. The method according to 41, wherein the detectable amount is        less than 100 copies of the target nucleic acid.    -   43. The method according to 41 or 42, wherein the C—(NH₂)₂NH⁺        salt in the lysis mixture is a guanidine salt.    -   44. The method according to any of 41 through 43, wherein the        guanidine salt is present in a 10× lysis buffer.    -   45. The method according to any of 41 through 44, further        comprising increasing the sensitivity of detection by        determining whether the sample contains the target nucleic acid        using a dual wavelength spectrophotometer.    -   46. The method according to any of 41 through 45, wherein the        elevated temperature in (a) is 95° C. and the effective time is        5 minutes.    -   47. The method according to any of 41 through 46, wherein (a)        further comprises allowing the sample in the lysis mixture to        incubate at room temperature before treating with an elevated        temperature.    -   48. The method according to 47, wherein if the incubation time        at room temperature is at least 30 minutes then the elevated        temperature may be 75° C. or less, for a period of time that is        60 minutes or less.    -   49. The method according to any of 41 through 48, wherein the        lysis mixture further comprises a detergent.    -   50. The method according to 49, wherein the detergent is Triton        X.    -   51. A composition comprising: a guanidine salt, a reducing agent        and detergent.    -   52. The composition according to 51, further comprising LiCl.    -   53. The composition according to 51 or 52, wherein the guanidine        salt is guanidine hydrochloride (Guanidine HCl).    -   54. The composition according to any of 51 through 53, wherein        the reducing agent is Triton X.    -   55. The composition according to any of 51 through 54, wherein        the reducing agent is Tris(2-carboxyethyl)phosphine        hydrochloride (TCEP).    -   56. The composition according to any of 51 through 55, further        comprising a biological sample.    -   57. The composition according to any of 51 through 56, wherein        the biological sample is saliva.    -   58. The composition according to any of 51 through 57, wherein        the composition further comprises a master mix according to any        of 1 through 20.    -   59. The composition according to any of 51 through 58,        comprising at least one LAMP primer set.    -   60. The composition according to any of 51 through 59, wherein        the at least one LAMP primer set is at least two different LAMP        primer sets for detecting a single target.    -   61. The composition according to any of 51 through 60, wherein        the composition further comprises a target polynucleotide.    -   62. The composition according to any of 51 through 61, wherein        the target polynucleotide is a coronavirus RNA.    -   63. A method for preparing a biological sample for an        amplification reaction; comprising:        -   (a) obtaining a lysis mixture comprising guanidine salt, a            reducing agent, and a detergent;        -   (b) combining the lysis mixture in (a) with a biological            sample containing a target polynucleotide to form a            diagnostic sample; and        -   (c) analyzing an aliquot of the diagnostic sample for            identifying if the template polynucleotide is present and            optionally the sequence of the template polynucleotide,            wherein the step of analyzing is selected from one or more            of (i) amplifying the target polynucleotide to detect the            presence of a template polynucleotide by a change in color            or fluorescence; (ii) amplifying the target polynucleotide            and sequencing the amplified polynucleotides; and (iii)            direct sequencing of an aliquot of the diagnostic sample.    -   64. A method according to 63, wherein a 2× lysis mixture        comprises guanidine salt in the range of 300 mM-1.5 M guanidine        hydrochloride, TritonX in the range of 1%-6%, and TCEP in the        range of 40 mM-120 mM.    -   65. The method of 64, wherein the 2× lysis mixture further        comprises LiCl.    -   66. The method of 65, wherein the LiCl is in the range of 50        mM-100 mM.    -   67. The method of any of 64 through 66, wherein the 2× lysis        mixture comprises 800 mM guanidine hydrochloride, 4% Triton X        and 80 mM TCEP.    -   68. The method of 67, wherein the 2× lysis mixture further        comprises 150 mM LiCl.    -   69. The method of any of 63 through 68, wherein (b) further        comprises increasing the temperature of the diagnostic sample to        release the polynucleotides from biological material in the        sample.    -   70. The method of 69, wherein the step of increasing the        temperature further comprises raising the temperature in the        range of 65° C.-95° C. wherein the time of incubation at the        raised temperature is inversely proportional to the temperature.    -   71. The method of 69, wherein increasing the temperature further        comprises raising the temperature to 75° C. for 20 minutes.    -   72. The method of 70, further comprises incubating the sample at        room temperature for 30 minutes prior to increasing the        temperature.    -   73. The method according to any of 63 through 72, wherein the        step of amplifying further comprises amplifying the target        polynucleotide by LAMP.    -   74. The method according to 73, wherein the LAMP is pH-dependent        colorimetric LAMP.    -   75. The method according to 74, wherein the pH of the lysis        mixture is at least pH 7.9 if the biological sample is saliva.    -   76. The method according to 63, wherein sequencing further        comprises adding sample barcodes for large scale multiplexing of        samples, wherein the bar code is added in the primer where        amplification precedes sequencing or by ligation when the target        polynucleotides are sequenced directly.    -   77. The method according to any of 40 through 48, and 63 through        75, further comprising a high throughput automated workflow for        performing the steps of the method.    -   78. A kit comprising the master mix of any of 1 through 20,        and/or the compositions of 32 through 39, and 51 through 62,        wherein the kit optionally further comprises a heating block        suitable for heating a reaction tube, plate, or paper, or a        plurality of the same.    -   79. A kit for performing multiplex loop mediated amplification        (LAMP) reaction, comprising:        -   (a) a plurality of sets of oligonucleotide primers, wherein            the different sets of primers hybridize to different            template sequences in the same target nucleic acid and are            suitable for amplifying the different template sequences of            the target nucleic acid by LAMP;        -   (b) a strand-displacing DNA polymerase; and        -   (c) a guanidinium salt;        -   wherein the reagents of (a)-(c) are in separate containers.    -   80. The kit according to 79, wherein the stand-displacing DNA        polymerase of (b) is contained in a master mix that further        comprises:        -   deoxynucleotide triphosphates including dUTP;        -   a reversible inhibitor of polymerase activity that blocks            polymerase activity at a temperature below 40° C.; and        -   at least one dye or fluorescent indicator for detecting an            amplification product by a change in color or fluorescence.    -   81. The kit according to 80, further comprising instructions to        combine the reagents of (a)-(c) with a target DNA in order to        amplify the different template sequences by LAMP.    -   82. The kit according to 81, further comprising a reducing        agent.    -   83. The kit according to 82, wherein the reducing agent is        Tris(2-carboxyethyl)phosphine hydrochloride (TCEP).    -   84. The kit according to 79, wherein the guanidinium salt is        guanidine hydrochloride.    -   85. The kit according to 80, wherein the indicator is a        metallo-chromatic dye.    -   86. The kit according to 85, wherein the metallo-chromatic dye        is hydroxynaphthol blue.    -   87. The kit according to 80, wherein the indicator is phenol        red.    -   88. The kit according to 80, wherein the master mix further        comprises a reverse transcriptase.    -   89. The kit of 79, wherein each set of oligonucleotide primers        comprises 4, 5 or 6 primers.    -   90. The kit according to 79, wherein the target nucleic acid is        derived from a coronavirus RNA and each set of primers        hybridizes to a different template sequence in the coronavirus        RNA genome, or cDNA copy thereof.    -   91. The kit according to 79, wherein the plurality of sets of        oligonucleotide primers comprise a mixture of 12 different        primers.    -   92. The kit according to 79, wherein the oligonucleotide primers        target multiple target nucleic acids with a plurality of sets of        oligonucleotide primers for each target.    -   93. A method comprising:        -   (a) combining the kit of 79 with a target nucleic acid to            produce a reaction mix; and        -   (b) incubating the reaction mix to produce an amplification            product.    -   94. The method according to 93, wherein the composition further        comprises a reverse transcriptase if the target nucleic acid is        RNA, and the method comprises reverse transcribing the RNA into        DNA.    -   95. The method according to claim 94, wherein the composition of        claim 79 further comprises at least one additional reagent        selected from the group consisting of: reverse transcriptase,        UDG, dUTP, dATP, dTTP, dGTP, dCTP, a color or fluorescent        indicator that changes color or fluorescence when amplification        has occurred, a reducing agent and a detergent.    -   96. The method according to 93, wherein the target nucleic acid        is from an infectious agent.    -   97. The method according to 96, wherein the infectious agent is        a coronavirus.    -   98. The method according to 93, wherein each set of        oligonucleotide primers comprises 4 or 6 primers.    -   99. The method of claim 93, wherein the plurality of sets of        primers for the target nucleic acid comprise at least 2 sets of        primers, each set of primers hybridizing to a different target        sequence in the coronavirus genome, or cDNA copy thereof.    -   100. The method of 93, wherein the plurality of sets of primers        for the target nucleic acid comprise at least 2 sets of primers        and (a) further comprises including multiple different target        nucleic acids wherein a plurality of sets of primers hybridize        to a different target sequence in each target nucleic acid.    -   101. A method for detecting SARS-CoV-2 virions in saliva of a        subject, comprising:        -   (a) collecting saliva in a receiving vessel containing            reagents comprising (i) oligonucleotides immobilized on a            substrate in a diluting volume of buffer containing DNase            inhibitors, RNase inhibitors and proteolytic agents or (ii)            lyophilized oligonucleotide immobilized on a substrate,            DNase inhibitors, RNase inhibitors and one or more            proteolytic agents for rehydration in contact with the            saliva;        -   (b) permitting virions in contact with the reagents to be            disrupted, releasing viral RNA for binding to the            immobilized oligonucleotides;        -   (c) removing the non-bound material from the receiving tube;        -   (d) adding a master mix containing primers specific for            SARS-CoV-2 RNA and/or cDNA for reverse transcribing the RNA            and amplifying the cDNA; and        -   (e) detecting a change in a signal resulting from amplifying            cDNA.    -   102. The method according to 101, wherein the oligonucleotides        are immobilized on beads or on the surface of the receiving        vessel.    -   103. The method according to 101 or 102, wherein the        oligonucleotides are characterized by a single sequence capable        of binding to either end of the virion RNA or to an internal        sequence in the virion RNA.    -   104. The method according to 101 or 102, wherein the        oligonucleotides are characterized by a plurality of different        sequences wherein each sequence targets a different region of        the viral genome.    -   105. The method according to 101 or 102, wherein the        oligonucleotides are characterized by one or a plurality of        sequences capable of acting as a primer to initiate reverse        transcription.    -   106. The method according to 101 or 102, wherein the        oligonucleotides are characterized by a plurality of sequences        capable of targeting different viral RNA genomes such as        SARS-CoV-2 and Influenza.    -   107. The method according to any of 101 through 106, wherein the        one or more proteolytic reagents are selected from Proteinase K,        thermolabile Proteinase K, salt, detergent, and reducing agent.    -   108. The method according to any of 101 through 107, wherein the        receiving vessel further contains a guanidinium salt.    -   109. The method according to any of 101 through 108, wherein        amplifying cDNA is achieved by isothermal amplification.    -   110. The method according to 109 wherein the isothermal        amplification is LAMP.    -   111. The method according to any of 101 through 107, wherein the        amplification is achieved by a polymerase chain reaction.    -   112. The method according to any of 101 through 111, wherein the        change in signal is a change in color or fluorescence.    -   113. The method according to 112, further comprising, detecting        the change in color or fluorescence by eye.    -   114. The method according to 112, further comprising, detecting        the change in color or fluorescence by a device.    -   115. The method according to any of 101 through 114, wherein the        RNAse inhibitors are aptamers or antibodies.    -   116. The method according to any of 101 through 115, wherein the        buffered reagents or the lyophilized reagents contain reagents        for facilitating hybridization of complementary single strand        nucleic acids.    -   117. The method according to any of 101 through 116, wherein the        one or more oligonucleotides contains a sample bar code.    -   118. The method according to 117, further comprising a pooling        step between (c) and (d) wherein multiple samples may be pooled        prior to reverse transcription or after reverse transcription        and prior to amplification of the cDNA.    -   119. A composition comprising a receiving vessel containing        reagents comprising (i) oligonucleotides immobilized on a        substrate in a diluting volume of buffer containing DNase        inhibitors, RNase inhibitors and proteolytic agents or (ii)        lyophilized oligonucleotide immobilized on a substrate, DNase        inhibitors, RNase inhibitors and one or more proteolytic agents        for rehydration in contact with the saliva; wherein the        oligonucleotides are characterized by one or more sequences,        wherein at least one sequence is complementary to a portion of        SARS-CoV-2 RNA.    -   120. The composition according to 119, wherein the        oligonucleotides are capable of hybridizing to at least one of        the polyA tail of the viral RNA, the capped end of the RNA or to        an internal target sequence.    -   121. The composition according to 119 or 120, wherein the        oligonucleotide is a primer for reverse transcription of the        hybridized virion RNA.    -   122. The composition according to any of 119 through 121,        wherein the one or more oligonucleotide contains modified        nucleotides.    -   123. The composition according to any of 119 through 122,        wherein the one or more oligonucleotides contain a sample        barcode.    -   124. The composition according to any of 119 through 123,        wherein the reagents further comprise one or more of the        following: Proteinase K, thermolabile Proteinase K, aptamer        inhibitors of RNAses and/or DNases, antibody inhibitors of        RNAses and/or DNases, salts to facilitate hybridization and/or        protein denaturation, detergents, surfactants and/or reducing        agents.    -   125. A kit comprising the compositions of 119 through 124.    -   126. A composition, comprising a mixture of LAMP primer sets        wherein each of a plurality of different primer sets in the        mixture have a specificity for a different respiratory virus.    -   127. A composition according to 126, further comprising a primer        set in the mixture having a specificity for a cellular non-viral        target.    -   128. The composition according to any of 126 or 127, wherein the        plurality of primer sets in the mixture are specific for one or        more target pathogens selected from a coronavirus, a rhinovirus,        a respiratory syncytial virus, an influenza virus, a        parainfluenza virus, a metapneumovirus, an adenovirus and a        bocavirus.    -   129. The composition according to any of 126 through 128,        wherein the mixture further comprises a plurality of duplex        oligonucleotides, wherein (i) each duplex is formed from a        5′-modified version of an FIP primer (Q-FIP) annealed to a        complementary oligonucleotide; (ii) the Q-FIP contains a        quenching molecule or a fluorescent label such that the        fluorescent label is quenched until one strand of the duplex        hybridizes to a target; and (iii) the fluorescent label for each        primer set can be differentiated by wavelength emission when        unquenched.    -   130. The composition according to any of 126 through 129,        wherein the concentration-of the primer sets in the mixture are        similar except for the primer set in the mixture that is        specific for the cellular non-viral target.    -   131. The composition according to any of 126 through 130,        wherein the concentration of the primer set in the mixture that        is specific for the cellular non-viral target has a        concentration in the range of 25%-80% for example 50-75% of the        primer sets for respiratory viruses.    -   132. A kit, comprising:        -   (a) a sample collection container;        -   (b) a mixture of primer sets according to any of 126 through            131, wherein the mixture may be in the sample collection            container or separate from the sample collection container;        -   (c) a polymerase and optionally a reverse transcriptase in            the same container as (a) or separate container; and        -   (d) instructions for performing multiplex loop mediated            amplification using a fluorescent probe and quencher.    -   133. A kit according to 132, wherein the sample collection        container contains a poloxamer surfactant.    -   134. A composition, comprising a biological sample combined with        a buffer containing an antifoaming agent, wherein the        antifoaming agent is a poloxamer.    -   135. A composition according to 134, further comprising one or        more LAMP primer sets.    -   136. A composition according to 134 or 135, wherein the        poloxamer is PF68.    -   137. A composition according to any of 134 through 136, wherein        the buffer further comprises TCEP and EDTA.    -   138. A kit comprising, a reaction container for receiving a swab        containing a biological sample or a liquid biological fluid, the        reaction container optionally containing a buffer containing a        poloxamer or wherein the buffer containing the poloxamer is        contained in a distinct compartment in the reaction container or        is provided in a separate tube for receiving an aliquot of the        biological sample.    -   139. A method, comprising:        -   (a) combining a biological sample from a nasal or buccal            swab or from saliva from a vertebrate subject, with a buffer            containing a poloxamer in a container;        -   (b) heating the biological sample in the container from (a)            to 95° C.; and        -   (c) allowing the sample to cool and adding one or more LAMP            primer sets.    -   140. The method according to claim 139, wherein the poloxamer is        PF68.    -   141. The method according to 139 or 140, further comprising:        -   (d) combining a plurality of LAMP primer sets with an            aliquot of the cooled sample in a LAMP reaction buffer where            each primer set comprises a quenched fluorescent dye in a            duplex oligonucleotide.    -   142. The method according to 141, wherein the plurality of LAMP        primer sets are specific for one or more virus nucleic acids        selected from the group consisting of a coronavirus, a        rhinovirus, a respiratory syncytial virus, an influenza virus, a        parainfluenza virus, a metapneumovirus, an adenovirus and a        bocavirus.    -   143. The method according to any of 139 through 142, wherein the        vertebrate subject is a mammal.    -   144. The method according to 143, further comprising:        -   (e) performing multiplex DARQ LAMP to determine the presence            of SARS-CoV-2 and/or Influenza virus in the subject.    -   145. The method according to any of 139 through 144, wherein the        LAMP reaction buffer contains dUTP for carryover prevention.    -   146. The method according to any of 139 through 145, wherein the        container is a well in a 96 well or 384 well dish or the        container is a tube.    -   147. The method according to any of 141 through 146, further        comprising:        -   (f) unquenching of the fluorescent dye in the presence of a            target nucleic acid to provide a fluorescent signal; and        -   (g) detecting the fluorescent signal by determining the            wavelength of emitted light for each fluorescent signal.    -   148. A kit for Loop-Mediated Isothermal Amplification (LAMP),        comprising:        -   (a) a strand displacing polymerase capable of copying DNA at            a temperature in the range of 50° C.-68° C.;        -   (b) a reversible inhibitor of the polymerase, wherein the            inhibitor inhibits the strand displacing DNA polymerase at a            temperature of below 50° C.;        -   (c) a thermolabile uracil DNA glycosylase (UDG) that is            inactivated at a temperature of above 50° C.;        -   (d) nucleoside triphosphates comprising dATP, dGTP, dCTP,            and dTTP; and dUTP; and

at least one indicator reagent that changes color or providesfluorescence if amplification occurs;

wherein any of (a) to (e) are separate or combined into one or moremixtures in the kit.

-   -   149. The kit according to 148, wherein the strand displacing        polymerase is a mesophilic bacterial strand displacing        polymerase and the temperature for copying the DNA is 65° C. for        less than 1 hour.    -   150. The kit according to 148 or 149, wherein the reversible        inhibitor is an oligonucleotide.    -   151. The kit according to any of the preceding embodiments,        further comprising a receiving container for a biological        sample.    -   152. The kit according to any of the preceding embodiments,        further comprising lysis reagents for adding to or contained in        the receiving container for lysing a biological sample to        release any target nucleic acids therein for amplification by        Loop-Mediated Isothermal Amplification (LAMP).    -   153. The kit according to 152, wherein the lysis reagents        comprise a reducing agent and a metal chelator.    -   154. The kit according to 153, wherein the reducing agent is        Tris (2-carboxyethyl) phosphine hydrochloride (TCEP).    -   155. The kit according to any of 152 to 154, wherein the lysis        reagents comprise a salt of C—(NH₂)₂NH⁺.    -   156. The kit according to any of 152 to 155, wherein the lysis        reagents comprise a poloxamer.    -   157. The kit according to any of the preceding claims, further        comprising: in a separate container, at least one set of LAMP        primers.    -   158. The kit according to 157, wherein the at least one set of        Loop-Mediated Isothermal Amplification (LAMP) primers further        comprise a plurality of primer sets for amplifying a plurality        of target nucleic acids in the biological sample.    -   159. The kit according to 158, wherein the at least one set of        Loop-Mediated Isothermal Amplification (LAMP) primers further        comprise a plurality of primer sets for amplifying a nucleic        acid target from a single virus strain in the biological sample.    -   160. The kit according to any of claims 148-151 and 155-159,        wherein any or all of the lysis reagents and/or reagents for        Loop-Mediated Isothermal Amplification (LAMP) amplification        except for a salt of C—(NH₂)₂NH⁺, are lyophilized, freeze dried        or in a solution.    -   161. The kit according to any of 148-151 and 155-160, further        comprising a buffer with a buffering concentration of no more        than equivalent to 5 mM Tris buffer for use with the kit        components if the indicator in (e) is a pH dependent        colorimetric dye.    -   162. The kit according to 152, further comprising instructions        for use with a biological sample wherein the biological sample        is selected from a body fluid or tissue, an agricultural sample,        a food sample, a waste product, and a pathogen.    -   163. The kit according to claim 162, wherein the biological        sample is a body fluid or tissue selected from the group        consisting of mucous, urine, lymph, blood, saliva, feces,        sputum, sweat, semen and biopsy.    -   164. The kit according to any of the previous claims were in the        enzymes and/or oligonucleotides and/or Loop-Mediated Isothermal        Amplification (LAMP) primer sets are immobilized on a substrate        for reacting with a nucleic acid in the biological sample.    -   165. The kit according to any of 148-161 and 163-165, wherein        the indicator reagent is a metallochromic dye.    -   166. The kit according to any of 148-164, wherein the indicator        reagent is a pH sensitive colorimetric dye.    -   167. The kit according to any of claims 148-161 and 163-165,        wherein the indicator reagent is a fluorescent dye.    -   168. The kit according to any of claims 148-168, further        comprising a reverse transcriptase.    -   169. The kit according to claim 168, wherein the reverse        transcriptase is selected from a virus derived reverse        transcriptase, or from a Group II intron reverse transcriptase.    -   170. The kit according to 168-169, further comprising a        reversible inhibitor of the reverse transcriptase.    -   171. The kit according to 152, wherein the biological sample is        saliva or a nasal swab and the target nucleic acid is a single        target RNA virus genome.    -   172. The kit according to any of 151-171, wherein the receiving        container for the biological sample is a vessel with a lid, the        lid containing a solution of indicator reagent for release into        the reaction vessel after lysis of the biological sample or        after Loop-Mediated Isothermal Amplification (LAMP).    -   173. The kit according to any of 148-172, suitable for use in a        high sample throughput workflow that is partially or completely        automated and further comprises a recording device for storing        and/or reporting positive sample data after detection of a        change in color or fluorescence of the indicator resulting from        amplification of the target nucleic acid.    -   174. The kit according to any of claims 151-173, wherein the        receiving container is selected from paper, a microfluidic        device and a polymer surface.    -   175. A reaction mixture, comprising: a thermolabile uracil DNA        glycosylase (UDG), a strand displacing polymerase, a reversible        inhibitor of the polymerase, and a salt of C—(NH₂)₂NH⁺.    -   176. A lysis mixture for releasing an RNA from a biological        sample for detection by amplification and/or sequencing,        comprising a poloxamer, a reducing agent and a metal chelating        agent.    -   177. The lysis mixture of 29, further comprising a salt of        C—(NH₂)₂NH⁺.    -   178. A master mix comprising: a thermolabile uracil DNA        glycosylase (UDG), a strand displacing polymerase, a reversible        inhibitor of the polymerase, a reverse transcriptase and a        reversible inhibitor of the reverse transcriptase.    -   179. A method for amplifying any target nucleic acid in a        biological sample by Loop-Mediated Isothermal Amplification        (LAMP), comprising:        -   (a) combining the biological sample with a lysis reagent to            form a lysis mix;        -   (b) incubating the lysis mix at a temperature of at least            60° C. for a period of time in the range of 3 minutes to 45            minutes;        -   (c) combining an aliquot of the heat treated mix after            step (b) with amplification reagents comprising a strand            displacing polymerase, a reversible inhibitor of the            polymerase, a thermolabile uracil DNA glycosylase (UDG),            nucleoside triphosphates, and at least one set of LAMP            primers that hybridize to the target nucleic acid, to            produce a reaction mix; and        -   (d) incubating the reaction mix under amplification            conditions for LAMP to permit inactivation of the            thermolabile UDG and amplification of the target nucleic            acid.    -   180. The method of 179, wherein the lysis reagent in (a)        comprises at least one of a salt of C—(NH₂)₂NH⁺ and a poloxamer        to produce a lysis mix.    -   181. The method of 179 or 180, wherein the lysis reagent        comprises a reducing agent and a metal chelating reagent.    -   182. The method of 179 or 180, wherein (b) further comprises        incubating the lysis mix at 95° C. for 5 minutes.    -   183. The method of any of 179-182, wherein the amplification        reagents include a reverse transcriptase and a reversible        inhibitor of the reverse transcriptase.    -   184. The method according to any of 179-183, wherein any of the        reagents in the lysis mix and any of the amplification reagents        may be lyophilized prior to combining with the biological        sample.    -   185. The method according to any of 179-184, wherein any of the        reagents in the lysis mix and any of the amplification reagents        may be immobilized on a matrix.    -   186. The method of any of 179-185, further comprising (c)        further comprises an indicator reagent that changes color or        provides fluorescence if amplification occurs.    -   187. The method according to 186, further comprising (e)        detecting a change in color or fluorescence of the indicator        reagent corresponding to the amplification of the target nucleic        acid.    -   188. The method according to any of 179-187, wherein the        biological sample is saliva.    -   189. The method according to any of 179-188, wherein the target        nucleic acid is an RNA virus and the amplification reagents        include a reverse transcriptase and a reverse transcriptase        reversible inhibitor.    -   190. The method according to any of 179-189, wherein the RNA        virus is a coronavirus and the amplification reagents include        multiple sets of primers.    -   191. The method according to 190, wherein the multiple sets of        primers include a plurality of primers sets for amplifying at        least two different sequences in the coronavirus genome or cDNA        copy of the coronavirus genome.    -   192. The method according to 191, wherein the at least two        different sequences are at least a portion of Gene N and a        portion of Gene E in the coronavirus.    -   193. A method for analyzing a biological sample to determine the        presence of a target nucleic acid, comprising:        -   (a) combining the biological sample with a lysis reagent            comprising a reducing agent, a metal chelator and at least            one of a guanidine salt and a poloxamer to produce a lysis            mix; and        -   (b) determining the presence of the target nucleic acid by            selectively amplifying the target nucleic acid in a reaction            mix that comprises an aliquot of the lysis mix.    -   194. The method of 193, wherein the reaction mix comprises an        indicator reagent that changes color or provides fluorescence if        amplification occurs, and the method further comprises        determining whether the reaction mix contains the target nucleic        acid based on a change in color or fluorescence.    -   195. The method of 193-194, wherein (b) further comprises:        incubating the reaction mix under amplification conditions for        Loop-Mediated Isothermal Amplification (LAMP) to permit        amplification of the target nucleic acid.    -   196. A method for detecting an RNA virus in saliva or a nasal        swab of a subject, comprising:    -   (a) collecting saliva in a receiving container that comprises:        -   (i) substrate immobilized oligonucleotides for binding viral            RNA and a lysis reagent mix comprising two or more reagents            selected from a guanidinium salt, a poloxamer, a reducing            agent, a DNase inhibitor, an RNase inhibitor, a detergent, a            metal chelator and a proteolytic agent; or        -   (ii) lyophilized substrate immobilized oligonucleotides for            binding viral RNA, and/or one or more lyophilized reagents            contained in a lysis reagent mix, wherein the lysis reagent            mix comprises two or more reagents selected from a            poloxamer, a reducing agent, DNase inhibitor, an RNase            inhibitor, a detergent, a metal chelator and a proteolytic            agent, wherein the lyophilized reagents become rehydrated            when contacted by the collected saliva;    -   (b) incubating the receiving container after step (a) at an        effective temperature and time to release nucleic acid from any        coronaviruses in the saliva for binding to the immobilized        oligonucleotides;    -   (c) removing the lysis reagent mix from the receiving vessel        leaving the coronavirus genome bound to the immobilized        oligonucleotides on the substrate;    -   (d) adding amplification reagents to the substrate after step        (c), wherein amplification reagents comprise reverse        transcription reagents and DNA amplification reagents, to make a        reaction mix; and    -   (e) incubating the reaction mix under amplification conditions        to amplify a cDNA copy of at least a portion of the coronavirus        genome.    -   197. The method according to claim 196, wherein the receiving        container is selected from a paper substrate, a microfluidic        device or a polymer surface.    -   198. The method according to 196 or 197, wherein the lysis        reagent mix in (i) comprises three or more reagents selected        from a guanidinium salt, a poloxamer, a reducing agent, a DNase        inhibitor, an RNase inhibitor, a detergent, a metal chelator and        a proteolytic agent or in (ii) comprises three or more reagents        where at least one reagent is lyophilized that is selected from        a poloxamer, a reducing agent, DNase inhibitor, an RNase        inhibitor, a detergent, a metal chelator and a proteolytic        agent.    -   199. The method of 196 or 197, wherein the reaction mix of (d)        further comprises an indicator reagent that changes color or        provides fluorescence if amplification occurs, and wherein the        method further comprises detecting a change in a signal that        indicates the presence of coronavirus in the saliva of the        subject.    -   200. The method according to any of 196-199, comprising        amplification reagents for Loop-Mediated Isothermal        Amplification (LAMP).    -   201. The method of any of 196-200, wherein the amplification        reagents comprise the reagents in the kit according to claim 1.    -   202. The method according to 201, wherein the amplification        reagents further comprise at least two sets of Loop-Mediated        Isothermal Amplification (LAMP) primers that target different        regions of a coronavirus genome.    -   203. The method according to 202, wherein the targeted regions        comprise Gene E and Gene N on the coronavirus genome.    -   204. The method according to any of 196-203, wherein the        amplification reagents comprise Loop-Mediated Isothermal        Amplification (LAMP) primer sets targeting a second viral genome        that is not a coronavirus wherein the LAMP primer sets are        combined in a single reaction mix and wherein the LAMP primer        set for the coronavirus is linked to a colorimetric indicator        that changes color after amplification that is detectable at one        wavelength and a second LAMP primer set for amplifying a second        non-coronavirus nucleic acid, having a colorimetric indicator        that changes color after amplification that is detectable at a        second wavelength.    -   205. The method according to any of 196-204, wherein the        amplifying step is followed by sequencing of the amplified        nucleic acid.    -   206. A method for amplifying a target nucleic acid by        Loop-Mediated Isothermal Amplification (LAMP), comprising:        -   (a) combining in a mixture, a biological sample from a            mammalian subject with a buffer comprising a poloxamer;        -   (b) heating the mixture to a temperature of at least 65° C.            for an effective time to denature proteins in the biological            sample;        -   (c) allowing the sample to cool; and        -   (d) amplifying one or more nucleic acids from the mix by            LAMP.    -   207. The method of 206, wherein the biological sample is saliva,        a nasal swab or a buccal swab.    -   208. A kit for use in diagnostic detection of a target nucleic        acid and variants thereof having undefined mutations, obtained        from a cell or virus in a biological sample, the kit comprising:        -   (a) a lyophilized mixture of a strand displacing polymerase            and an indicator reagent and optionally a lyophilized            reverse transcriptase, wherein            -   (i) the indicator reagent is characterized by its                ability to change color or provide fluorescence in a                nucleic acid amplification reaction; and            -   (j) the strand displacing polymerase when rehydrated is                capable of amplifying DNA at a temperature in the range                of 50° C.-68° C.        -   (b) a universal primer set suitable for loop mediated            amplification (LAMP) of the target nucleic acids and            variants thereof having undefined mutations;    -   wherein any of the reagents in the kit may be combined in a        mixture in a single container or provided in separate        containers.    -   209. The kit according to 208 wherein the target nucleic acid is        a target DNA that is the reverse transcription product of an RNA        virus.    -   210. The kit according to 208 or 209, wherein the indicator        reagent is a molecular beacon.    -   211. The kit according to any of 208-210, wherein the universal        primer set suitable for LAMP is capable of hybridizing to the        target DNA in the presence of a plurality of undefined mutations        to provide a positive result for the target DNA in a        predetermined assay time period otherwise determined for a        positive sample of a target nucleic acid having a known        sequence.    -   212. The kit according to any of 208-211, further comprising (c)        a lysis reagent in a container for receiving the biological        sample, wherein the lysis reagents comprise a reducing agent and        a metal chelator.    -   213. The kit according to 212, wherein the reducing agent is        Tris (2-carboxyethyl) phosphine hydrochloride (TCEP).    -   214. The kit according to 212, wherein the lysis reagents        comprise at least one of a salt of C—(NH₂)₂NH⁺ and a poloxamer.    -   215. The kit according to any of 208-214 wherein one or more of        components in (a)-(b) are immobilized on a substrate.    -   216. The kit according to any of 208-215, wherein the indicator        reagent is a metallochromic dye.    -   217. The kit according to any of 208-216, comprising the reverse        transcriptase, wherein the reverse transcriptase is a virus        encoded reverse transcriptase, or a bacteria encoded intron II        reverse transcriptase.    -   218. A method for detecting a target nucleic acid or unknown        variant thereof in a biological sample by Loop-Mediated        Isothermal Amplification (LAMP), comprising:        -   (a) combining the biological sample with a lysis reagent to            form a lysis mix;        -   (b) incubating the lysis mix at a temperature of at least            60° C. for a period of time in the range of 2 minutes to 45            minutes;        -   (c) combining in a reaction mix, an aliquot of the heat            treated lysis mix of step (b) with amplification reagents            comprising a strand displacing polymerase, a reversible            inhibitor of the polymerase, nucleoside triphosphates, and            at least one set of LAMP primers that is capable of            hybridizing to the target nucleic acid and to undefined            variants thereof; and        -   (d) incubating the reaction mix for a reaction positive            period of time under amplification conditions for LAMP to            detect the presence of the target nucleic acid or undefined            variants thereof in the sample.    -   219. The method of 218, wherein the lysis reagent comprises a        reducing agent and a metal chelating reagent.    -   220. The method of 218 or 219, wherein (b) further comprises        incubating the lysis mix at 95° C. for 5 minutes.    -   221. The method of any of 218-220, wherein the amplification        reagents include a reverse transcriptase and a reversible        inhibitor of the reverse transcriptase.    -   222. The method according to any of 218-221, wherein any of the        lysis reagents and any of the amplification reagents may be        lyophilized prior to combining with the biological sample.    -   223. The method according to any of 218-223, wherein any of the        lysis reagents and any of the amplification reagents may be        immobilized on a matrix. 224. The method according to any of        218-223, wherein the biological sample is saliva. 225. The        method according to any of 218-223, wherein the target nucleic        acid is an RNA genome from a virus.    -   226. The method according to 225, wherein the virus is a        coronavirus and the amplification reagents include multiple sets        of primers.    -   227. The method according to any of 218-226, further comprising        step (e) sequencing the detected target nucleic acid or variants        thereof to determine the presence of novel mutations.

EXAMPLES

All reagents are commercially available and provided by New EnglandBiolabs, Ipswich, Mass. unless otherwise specified. Although theexamples are provided for the coronavirus they are also applicable toother pathogens and to the analysis of DNA and RNA.

Example 1: Identification of SARS-CoV-2 Virus RNA

SARS-CoV-2 virus RNA is analyzed directly from nasal swabs using avisual, colorimetric detection. This simple and sensitive methodprovides an opportunity to facilitate virus detection in the fieldwithout a requirement for complex diagnostic infrastructure. The generalfeatures of the method were reported in Tanner, et al. BioTechniques58:59-68 (2015) and reagents for conducting the method are provided byNew England Biolabs (M1800). Here the sensitivity of the method wastested for the coronavirus described as SARS-CoV-2.

LAMP Primer Design and Testing

5 sets of LAMP oligonucleotide primers targeting two fragments (Table 1)of SARS-CoV-2 sequence (GenBank accession number MN908947) were designedusing the online software Primer Explorer V5 (available for free use at:https://primerexplorer.jp/e/). The two fragments corresponded to the 5′region of the ORF1a gene and Gene N. Three sets of primers were designedto target ORF1 and two for GeneN. Each set of primers was tested withsynthetic DNA substrates (gBlocks®, Integrated DNA Technologies,Coralville, Iowa) and RNA (in vitro transcribed RNA from that DNA) priorto clinical use.

TABLE 1 Sequences of amplicons and LAMP primers LAMP primer or AmpliconSequence ORF1a CCCTATGTGTTCATCAAAC GTTCGGATGCTCGAACTGCACCTCATGGTCATGTTATG GTTGA (SEQ ID NO: 1) Fragment GCTGGTAGCAGAACTCGAAGGCATTCAGTACGGTCGTA GTGGTGAGACACTTGGTGT CCTT (SEQ ID NO: 2)GTCCCTCATGTGGGCGAAA TACCAGTGGCTTACCGCAA GGTTCTTCTTCGTAAGAACGGTA (SEQ ID NO: 3) ATAAAGGAGCTGGTGGCCA TAGTTACGGCGCCGATCTAAAGTCATTTGACTTAGGCG ACGA (SEQ ID NO: 4) GCTTGGCACTGATCCTTAT GAAGA(SEQ ID NO: 5) ORF1a-A ORF1a-A-F3 CTGCACCTCATGGTCATGT T (SEQ ID NO: 6)ORF1a-A-B3 AGCTCGTCGCCTAAGTCA A (SEQ ID NO: 7) ORF1a-A-FIPGAGGGACAAGGACACCAAG TGTATGGTTGAGCTGGTAG CAGA (SEQ ID NO: 8) ORF1a-A-BIPCCAGTGGCTTACCGCAAGG TTTTAGATCGGCGCCGTAA C (SEQ ID NO: 9) ORF1a-A-LFCCGTACTGAATGCCTTCGA GT (SEQ ID NO: 10) ORF1a-A-LB TTCGTAAGAACGGTAATAAAGGAGC (SEQ ID NO: 11) ORF1a-B ORF1a-B-F3 TCATCAAACGTTCGGATGC T(SEQ ID NO: 12) ORF1a-B-B3 TATGGCCACCAGCTCCTT (SEQ ID NO: 13)ORF1a-B-FIP CGACCGTACTGAATGCCTT CGAGAACTGCACCTCATGG TCAT (SEQ ID NO: 14)ORF1a-B-BIP AGACACTTGGTGTCCTTGT CCCAGAAGAACCTTGCGGT AAGC (SEQ ID NO: 15)ORF1a-B-LF CTGCTACCAGCTCAACCAT AAC (SEQ ID NO: 16) ORF1a-B-LBTCATGTGGGCGAAATACCA GT (SEQ ID NO: 17) ORF1a-C ORF1a-C-F3CTGCACCTCATGGTCATGT T (SEQ ID NO: 18) ORF1a-C-B3 GATCAGTGCCAAGCTCGTC(SEQ ID NO: 19) ORF1a-C-FIP GAGGGACAAGGACACCAAG TGTGGTAGCAGAACTCGAA GGC(SEQ ID NO: 20) ORF1a-C-BIP CCAGTGGCTTACCGCAAGG TTTTAGATCGGCGCCGTAA C(SEQ ID NO: 21) ORF1a-C-LF ACCACTACGACCGTACTGA AT (SEQ ID NO: 22)ORF1a-C-LB TTCGTAAGAACGGTAATAA AGGAGC (SEQ ID NO: 23) Gene NATGACCAAATTGGCTACTA CCGAAGAGCTACCAGACGA ATTCGTGGTGGTGACGGTA AAAT(SEQ ID NO: 24) fragment GAAAGATCTCAGTCCAAGA TGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGG ACTT (SEQ ID NO: 25) CCCTATGGTGCTAACAAAGACGGCATCATATGGGTTGC AACTGAGGGAGCCTTGAAT ACAC (SEQ ID NO: 26)CAAAAGATCACATTGGCAC CCGCAATCCTGCTAACAAT GCTGCAATCGTGCTAC (SEQ ID NO: 27)Gene N-A GeneN-A-F3 TGGCTACTACCGAAGAGCT (SEQ ID NO: 28) GeneN-A-B3TGCAGCATTGTTAGCAGGA T (SEQ ID NO: 29) GeneN-A-FIP TCTGGCCCAGTTCCTAGGTAGTCCAGACGAATTCGTGG TGG (SEQ ID NO: 30) GeneN-A-BIP AGACGGCATCATATGGGTTGCACGGGTGCCAATGTGAT CT (SEQ ID NO: 31) GeneN-A-LF GGACTGAGATCTTTCATTTTACCGT (SEQ ID NO: 32) GeneN-A-LB ACTGAGGGAGCCTTGAATA CA (SEQ ID NO: 33)Gene N-B GeneN-B-F3 ACCGAAGAGCTACCAGACG (SEQ ID NO: 34) GeneN-B-B3TGCAGCATTGTTAGCAGGA T (SEQ ID NO: 35) GeneN-B-FIP TCTGGCCCAGTTCCTAGGTAGTTCGTGGTGGTGACGGT AA (SEQ ID NO: 36) GeneN-B-BIP AGACGGCATCATATGGGTTGCACGGGTGCCAATGTGAT CT (SEQ ID NO: 37) GeneN-B-LF CCATCTTGGACTGAGATCTTTCATT (SEQ ID NO: 38) GeneN-B-LB ACTGAGGGAGCCTTGAATA CA (SEQ ID NO: 39)

These primers in a colorimetric LAMP assay were first tested onsynthetic sequences corresponding to regions of the SARS-CoV-2 either asDNA or as RNA.

DNA fragments containing two SARS-CoV-2 sequences were synthesized asgBlocks. T7 RNA polymerase promoter sequences were added by PCR (M0493)(numbers indicate New England Biolabs, Inc. catalog ID numbers unlessotherwise noted). The PCR reaction utilized primer pairs where oneprimer of the pair containing the T7 RNA promoter sequence. The PCRamplicon was then transcribed by in vitro transcription (E2050) toproduce RNA with sequences that mimicked the selected portions of theSARS-CoV-2 virus. This RNA was purified using RNA clean up columns(T2040). The resulting RNAs as well as the corresponding gBlocks DNAwere serially diluted in 10-fold increments using 0.1×TE buffercontaining 0.01% Tween 20.

RT-LAMP reactions (for RNA) and LAMP (for DNA) using fluorescent dyeswere performed using WarmStart Colorimetric LAMP 2× master mix (for DNA& RNA) (NEB product M1800) supplemented with 1 μM SYTO™-9 fluorescentdouble-stranded DNA binding dye (Thermo Fisher Scientific, Waltham,Mass. (S34854)) and incubated on a real-time qPCR machine (CFX96)Bio-Rad, Hercules, Calif.)) for 120 cycles with 15 seconds each cycle(total ˜40 minutes). This was performed to measure amplification in realtime continuously over a 40 minute time period. This was done to confirmthat amplification corresponded to color change and provide correlationsbetween input and color change.

The colorimetric LAMP assay was first described in U.S. Pat. Nos.9,034,606, 9,580,748 and US 2019/0169683 herein incorporated byreference. A weak buffer was described for use in the assay asdescribed.

All LAMP assays were performed in a 20 μl reaction mixture containing 2μL of 10× primer mix of 16 μM (each) of Forward Inner Primer (FIP) andBackward Inner Primer (BIP), 2 μM (each) of F3 and B3 primers, 4 μM(each) of Forward Loop (LF) and Backward Loop (LB) primers, 10 μL ofWarmStart Colorimetric Lamp 2× master mix (M1800) 5 μL of DNAse, RNasefree water and 3 μl of RNA template. Individual primer pair sets thatwere optimal (where one set includes 6 primers) were selected for ORF1and Gene N.

TABLE 2 Primer 10X concentration 1X concentration FIP 16 μM 1.6 μM BIP16 μM 1.6 μM F3  2 μM 0.2 μM B3  2 μM 0.2 μM Loop F  4 μM 0.4 μM Loop B 4 μM 0.4 μM

TABLE 3 No Template RNA target control DNA target detection (NTC)WarmStart 12.5 μl   12.5 μl   12.5 μl   Colorimetric LAMP 2X master mixLAMP Primer Mix (10X) 2.5 μl  2.5 μl  2.5 μl  Target DNA  1 μl — —Target RNA —  1 μl — dH₂O  9 μl  9 μl 10 μl Total volume 25 μl 25 μl 25μl Note: Make primer stock in molecular biology grade H₂O rather than TEor other buffer in order to avoid carryover of additional buffer to theLAMP reaction. Prepare primer stocks in nuclease free water and store at−20° C. for up to 2 years.

Instructions from the New England Biolabs website (www.neb.com) weregenerally followed unless stated otherwise: 24 μl of the 2× master mix,plus primers and dH₂O were added into each desired reaction vessels and1 μl of sample was added. After mixing, the reaction solutions wereconfirmed to have a bright pink color, indicating an initial high pHrequired for successful pH-LAMP reaction. The reaction mixture wasincubated at 65° C. for 30 minutes. The tubes or vessels were thenexamined by eye to determine positive reactions that turned yellow ornegative reactions that remained pink. Reactions can be examined earlierif desired. High copy or input reactions can exhibit full color changein as little as 10-15 minutes after incubation at 65° C. The color wasvisible directly on removal from the incubation temperature and could beintensified by allowing reaction to cool to room temperature. The resultwere photographed or scanned to record the colorimetric results, orsimply kept at room temperature in the reaction vessel.

For Identification of SARS-CoV-2 virus RNA in test samples anddetermining the sensitivity of the assay, positive control test sampleswere prepared as follows: synthetic viral RNAs were spiked into Helacells, which were then diluted and lysed using Luna Cell Ready LysisModule (E3032). Each lysate was then diluted 10× with 0.1×TE+0.01% Tween20 and 1 μL was added to standard colorimetric LAMP reactions.

For compatibility with blood recovery, the synthetic RNA described abovewas spiked into 200 μL whole human blood (Quadrant Health Strategies,Beverly, Mass.) and then purified the total blood RNA using MonarchTotal RNA Miniprep Kit (T2010).

The results of pH dependent detection sensitivity assays showed that allfive primer sets provided similar detection sensitivity and couldconsistently detect as low as 120 copies of the viral RNA (or 4.8copies/pi) as determined by serial dilution of ˜120 million copies downto ˜120 copies (per 25 μL reaction) at 10-fold intervals in thecolorimetric LAMP reactions. The relative efficiency of pH dependentcolorimetric LAMP using RNA or DNA templates was determined from thereal time LAMP signal using synthetic RNA with similarly diluted gBlockdsDNA. For the 2 primer sets we compared, one showed slightly sloweramplification and detection with RNA template while the other appearedslightly faster, confirming the RNA is efficiently converted to cDNA bythe reverse transcriptase (WarmStart RTx) and subsequently amplified viaLAMP by the DNA-dependent DNA polymerase (Bst 2.0 WarmStart). Thisresult was not adversely affected by the presence of UDP in the mastermix to prevent carryover.

Example 2: Analysis of Total RNA from Crude Lysates for Identificationof SARS-CoV-2 (COVID-19) Virus RNA

Crude cell lysate was used in order to avoid an RNA purification step.The results indicated that about 480 copies were detected with four ofthe five primer sets tested in Example 1, showing a similar sensitivityas the detection sensitivity with synthetic RNA alone (FIG. 3A) with nointerference by the lysate to either the amplification efficiency orvisual color change. A mock experiment was set up during purification oftotal RNA to determine whether the synthetic RNA spiked into biologicalsample could be recovered. Various amounts of synthetic RNA were spikedinto whole human blood and total blood RNA was purified. We were able torecover and detect the spiked RNA (FIG. 3B), indicating the total RNAdid not cause detectable interference during the purification or thedetection process. While the column-based approach is less compatiblewith the simple, field detection enabled by colorimetric LAMP, this is atypical laboratory workflow and can be used with simple isothermalamplification in a similar fashion to more expensive and involved qPCRdetection workflows.

Example 3: Nucleic Acid Carryover Prevention

Using the colorimetric LAMP assay described in Example 1, the benefit ofusing a thermolabile UDG and a 50:50 dTTP: dUTP in addition to dCTP,dGTP and dATP was demonstrated. FIG. 6A shows that this change inreagents did not affect the sensitivity or the specificity of the assay.Moreover, these additions to the master mix were effective in removingcarryover nucleic acids from one sample to the next.

More specifically: two Carryover Prevention WarmStart Colorimetric LAMP2× master mixes (abbreviated CP-LAMP MM) were developed and evaluated inan RT-LAMP functional assay, to determine whether the carryoveradditives interfered with the detection reaction as follows:

Each RT-LAMP reaction contained 1×CP-LAMP MM (no UDG or with UDG),1×LAMP primers, genomic RNA, and 1×LAMP fluorescent dye in a reactionvolume of 25 μL. High-copy reactions (n=3) contained 10 ng genomic RNA;low-copy reactions (n=6) contain 0.3 ng genomic RNA; and no-template(NT) reactions (n=1) contain no RNA. The plate was incubated at 65° C.for 75 minutes in a qPCR instrument, then imaged in a flatbedfluorescence scanner. The results are shown in FIG. 6A. A control wasadded as shown which had neither dUTP or UDG. No difference was observedbetween control and samples with the carryover prevention additives.

Carryover prevention was effective as demonstrated in FIG. 6B.

Each RT-LAMP reaction contains 1×CP-LAMP MM (with UDG), 1×LAMP primers,genomic RNA, and 1×LAMP fluorescent dye in a reaction volume of 25 μl.Both rows are identical replicates. The first well in each row containsapproximately 1 ng (1000 pg) of genomic RNA. From the second wellonwards, 10-fold dilutions were completed, with the last well in eachrow serving as a no-template (NT) reaction with no RNA. The plate wasincubated at 65° C. for 75 minutes in a qPCR instrument, then imaged ina flatbed fluorescence scanner. No amplified product was observed inamounts where carryover occurs.

Example 4: Nasal Sampling for Detecting SARS-CoV-2

Nasal samples are collected by swab and placed in sterile water in amicrofuge tube. An aliquot of the sample is then combined with a mastermix prepared as described above (see Example 3). Thermolabile UDG (NewEngland Biolabs, Ipswich, Mass.) is added according to themanufacturer's instructions. Four primer sets from Table 1 described inExample 2 can be used here although a single set of primers for each ofORF1 and GeneN is sufficient. Modifications of the primers described inTable 1 can also be utilized. Other regions in the virus may beadditionally or alternatively utilized. The reaction mixture is thenheated to a temperature of about 65° C. using a temperature block for15-60 minutes at which time the amplification is complete. The color ofthe reaction is then reviewed to reveal the presence or absence of thetarget nucleic acid. The entire reaction is amenable to substantialscaling up and can be executed in less than 1 hour from collection ofsample to receiving the results.

Example 5: Colorimetric LAMP Using Lyophilized Colorimetric LAMP Mix

2× master mix (2×MM) for colorimetric LAMP was prepared using standardconcentrations of LAMP reaction components described above (Bst 2.0 andRTx enzymes, aptamers to both, nucleotides, pH dependent dye, salt,detergent in a weakly buffered solution) together with 150 mM Trehalose,glycerol-free WarmStart RTx and high concentrations of WarmStart Bst2.0. Extra KOH was added to half of the mix to increase the pH of the2×MM to pH 8.2 from pH 8.0. To determine whether LAMP activity of the2×MM were the same at both pHs for lyophilized samples stored at roomtemperature and aqueous samples stored at −20° C., 12.5 μl of 2×MM at pH8.0 and 12.5 μl of the 2×MM at pH 8.2 were lyophilized and an equalvolume of 2×MM at the different pHs in liquid form were stored at −20°C. Lyophilization (freeze drying) was performed under standardconditions (see for example, Millrock Technologies, NY, LabogeneDenmark). The lyophilized 2×MMs were reconstituted with 12.5 μl H₂O andthe pH was measured. The pH in the reconstituted 2×MM was found to havedecreased by about 0.25 units. The reconstituted 2×MM and the 2×MMpreviously stored at −20° C. were then added to 12.5 μl ofprimer/template mix and 1 μM dsDNA binding fluorescent dye (Syto-9). Theprimer set for HMBS2 was used for RT-LAMP and the template was Jurkattotal RNA at 10 ng or 0.3 ng. The reaction was incubated in a Bio-RadIQ™5 Real Time PCR machine (Bio-Rad, Hercules, Calif.) to monitor thespeed of the reaction and colorimetric or fluorometric detection ofamplicons at the end of a 45 minute incubation at 65° C.

Results: both batches (initial pH 8.0 or pH 8.2, only the data from thepH 8.2 batch is shown) worked well after lyophilization. There was nodifference in colorimetric detection (FIG. 7, and FIG. 9A-9B) or realtime detection (FIG. 8A-8C and FIG. 9C) with either high or low amountof template in the RT-LAMP.

Example 6: PAR-Based Colorimetric Detection of Nucleic AcidAmplification is an Effective Alternative to pH Dependent ColorimetricLAMP

A standard 2×LAMP master mix was prepared (see Example 1) and added toDNA so that the reaction mix contained using the following DNApolymerases in separate reactions: Bst LF, Bst 3.0, Bst 2.0 or WarmStartBst 2.0 (all products from New England Biolabs, Ipswich, Mass.) instandard amplification buffer containing Tris-HCl, pH 8.8 at 25° C.;(NH₄)₂SO₄; KCl; MgSO₄. The buffer was varied from 0-4% Triton X-100. PARconcentration was varied from 250 μM to 50 μM PAR with results shown for150 μM, 100 μM, 75 μM and 50 μM (FIG. 13A-13D). MnCl₂ was usedthroughout at concentrations in the range of 0.4 mM-1.6 mM. In FIG.13A-13D, the reactions shown contained 0.5 μM MnCl₂. The LAMP primer setwas BRCA2b including FIP/BIP/F3/B3/LF/LB (see below). 1 μl Hela genomicDNA (100 ng/μl) was used in the positive samples and no DNA in thecontrols. The reaction was performed at 65° C. for 1 hour. A positiveendpoint was yellow corresponding to the reaction of manganese withpyrophosphate. The negative control was orange corresponding to thereaction of manganese with PAR.

TABLE 4 Primers for PAR-based colorimetric test BRCAb_F3TCCTTGAACTTTGGTCTCC (SEQ ID NO: 40) BRCAb_B3 CAGTTCATAAAGGAATTGA TAGC(SEQ ID NO: 41) BRCAb_FIP ATCCCCAGTCTGTGAAATT GGGCAAAATGCTGGGATTATAGATGT (SEQ ID NO: 42) BRCAb_BIP GCAGCAGAAAGATTATTAACTTGGGCAGTTGGTAAGTA AATGGAAGA (SEQ ID NO: 43) BRCAb_LFAGAACCAGAGGCCAGGCGAG (SEQ ID NO: 44) BRCAb_LB AGGCAGATAGGCTTAGACTC AA(SEQ ID NO: 45)

Example 7: Non-Ionic Detergent Increases the Positive Signal in a LAMPReaction Using PAR to Detect Sample

An example of a non-ionic detergent (Triton X-100) was added to the LAMP2× master mix containing PAR. In this example, the reaction mixcontained Bst 2.0, PAR (200 μM), Manganese (0.8 mM), Isothermalamplification buffer with 2% Triton X-100, the BRCA 2b primer set asused in Example 5 and 1 μl Hela gDNA. Although the beneficial effect ofadding Triton X-100 to the colorimetric PAR LAMP reaction is shown here,any non-ionic detergent from the Triton series or from the Brij seriesis expected to show similar benefits. The results are shown in FIG.11A-11B. 2% Triton X-100 was used in FIG. 14 to provide enhanced signalwithout adversely affecting polymerase activity although 1%-3% TritonX-100 also showed enhanced signal in visible wave lengths in FIG.13A-13D.

Example 8: Guanidine Hydrochloride Significantly Increases the Rate ofIsothermal Amplification Reactions

(a) LAMP

Guanidine hydrochloride (10 mM-60 mM) not only increased the rate of theLAMP reaction performed according to Example 1, but also improved thelimit of detection sensitivity (see FIG. 16, FIG. 17A-17C and FIG.18A-18D).

-   -   (i) BRCA and CFTR detection: Standard LAMP amplification was        performed in 1× ThermoPol buffer and Bst 2.0 DNA polymerase at        65° C. using 10 ng of genomic DNA isolated from Hela cells as        template. Two amplification targets were tested: BRCA gene        fragment and CFTR gene fragment. Guanidine hydrochloride at a        final concentration of 0 mM-60 mM were added to the reactions.        The amplification reactions contained 1 μM dsDNA binding        dye-Syto9 and the reaction was performed and the reaction speed        was monitored on a Bio-Rad IQ5 Real Time PCR machine.    -   (ii) SARS-CoV-2 detection: single primer set in a single LAMP        assay: As shown in FIG. 16, guanidine significantly increased        the LAMP reaction speed for both primer sets with optimal        concentration range between 20 mM-40 mM for primer sets 3 and 4        for coronavirus detection. The primer sets were tested against        an AccuPlex™ SARS-CoV-2 Verification Panel (SeraCare Milford,        Mass.) where the viral RNA is contained in a noninfectious viral        protein coat.    -   (iii) SARS-CoV-2 detection: multiple primer sets in a single        LAMP assay Multiple primers used in a single LAMP reaction        improved the sensitivity of colorimetric LAMP reactions. For        example, when primer set 3 and 4 were used together, sensitivity        of the LAMP assay increased (see FIG. 19A-19C).

Primer Set 1 or 5: As1e/Orf1a (5′-3′):

As1e_F3 CGGTGGACAAATTGTCAC (SEQ ID NO: 46) As1e_B3 CTTCTCTGGATTTAACACACTT (SEQ ID NO: 47) As1e_LF TTACAAGCTTAAAGAATGT CTGAACACT(SEQ ID NO :48) As1e_LB TTGAATTTAGGTGAAACAT TTGTCACG (SEQ ID NO: 49)As1e_FIP TCAGCACACAAAGCCAAAA ATTTATTTTTCTGTGCA AAGGAAATTAAGGAG(SEQ ID NO: 50) As1e_BIP TATTGGTGGAGCTAAACTT AAAGCCTTTTCTGTACAATCCCTTTGAGTG (SEQ ID NO: 51)

Primer Set 2: Gene N-A (5′-3′):

GeneN-F3 TGGCTACTACCGAAGAGCT (SEQ ID NO: 28) GeneN-B3TGCAGCATTGTTAGCAGGAT (SEQ ID NO: 29) GeneN-FIP TCTGGCCCAGTTCCTAGGTAGTCCAGACGAATTCGTGGTGG (SEQ ID NO: 30) GeneN-BIP AGACGGCATCATATGGGTTGCACGGGTGCCAATGTGATCT (SEQ ID NO: 31) GeneN-LoopF GGACTGAGATCTTTCATTTTACCGT (SEQ ID NO: 32) GeneN-LoopB ACTGAGGGAGCCTTGAATA CA(SEQ ID NO: 33)

Primer Set 3

Gene N-2 N2-F3 ACCAGGAACTAATCAGACAAG (SEQ ID NO: 52) N2-B3GACTTGATCTTTGAAATTTGG ATCT (SEQ ID NO: 53) N2-FIP TTCCGAAGAACGCTGAAGCG-GAACTGATTACAAACAT TGGCC (SEQ ID NO: 54) N2-BIP CGCATTGGCATGGAAGTCAC-AATTTGATGGCAC CTGTGTA (SEQ ID NO: 55) N2-LF GGGGGCAAATTGTGCAATTTG(SEQ ID NO: 56) N2-LB CTTCGGGAACGTGGTTGACC (SEQ ID NO: 57)

Primer Set 4

Gene E E1-F3 TGAGTACGAACTTATGTACTCAT (SEQ ID NO: 58) E1-B3TTCAGATTTTTAACACGAGAGT (SEQ ID NO: 59) E1-FIP ACCACGAAAGCAAGAAAAAGAAGTTCGTTTCGGAAGAGACAG (SEQ ID NO: 60) E1-BIP TTGCTAGTTACACTAGCCATCCTTAGGTTTTACAAGACTC ACGT (SEQ ID NO: 61) E1-LB GCGCTTCGATTGTGTGCGT(SEQ ID NO: 62) E1-LF CGCTATTAACTATTAACG (SEQ ID NO: 63)

Primer Set 1 or 5: As1e/Orf1a (5′-3′) SEQ ID NO: 46-51:

As1e_F3 CGGTGGACAAATTGTCAC (SEQ ID NO: 46) As1e_B3 CTTCTCTGGATTTAACACACTT (SEQ ID NO: 47) As1e_LF TTACAAGCTTAAAGAATG TCTGAACACT(SEQ ID NO :48) As1e_LB TTGAATTTAGGTGAAACAT TTGTCACG (SEQ ID NO: 49)As1e_FIP TCAGCACACAAAGCCAAAA ATTTATTTTTCTGTGCAA AGGAAATTAAGGAG(SEQ ID NO: 50) As1e_BIP TATTGGTGGAGCTAAACTT AAAGCCTTTTCTGTACAATCCCTTTGAGTG (SEQ ID NO: 51)

In addition, similar effect was observed with Bst DNA polymerase, largefragment, Bst 3.0, and in a RT-LAMP with RTx or AMV reversetranscriptase.

We also tested several related compounds containing the guanidine moiety(guanidinium compounds) and found that they also increased the rate ofLAMP. The compounds tested included Guanidine thiocyanate, Guanidinechloride and Guanidine sulfate (see FIG. 15).

The observed increase in rate of a LAMP reaction could be furtherenhanced in the presence of varying amounts of salt concentrations. LAMPreactions were set up in ThermoPol buffer (10 mM KCl) for Bst2.0 (seeFIG. 17B or in isothermal amplification buffer (50 mM KCl) for Bst 3.0(see FIG. 17C) using a lambda1 primer set with 0.5 ng lambda DNA with orwithout 30 mM guanidine hydrochloride. Addition of guanidine stimulatedthe LAMP amplification rate significantly at the lower end of the KClconcentration both with Bst 2.0 and Bst 3.0.

(b) Helicase-Dependent Amplification (HDA)

Standard HDA in IsoAmp II kit (H0110) was performed using 0.1 ng plasmidprovided in the kit but added guanidine hydrochloride at a finalconcentration of 0 mM-60 mM. The reactions were performed at 65° C. andEvaGreen dye was included to monitor the progression of amplification.The Tt (time to threshold) was used to estimate the rate ofamplification. Tt was shown to be 5 minutes shorter than standard HDAreactions. It was concluded that guanidine HCl increase melt temp for0.5° C. per 10 mM up to 60 mM guanidine hydrochloride. Isothermalreactions were monitored in the presence of reduced NaCl in the buffer.Increased rates were observed when NaCl was reduced from the standardamount of 40 mM to 10 mM NaCl. Less NaCl gave a higher RFU signal (seeFIG. 17A).

Example 9: Detection of Polynucleotides in Saliva Samples UsingpH-Dependent Colorimetric LAMP

Below is an example of the use of a lysis buffer suitable for directlyassaying saliva samples in a pH dependent colorimetric LAMP. Theworkflow from saliva collection to LAMP analysis is shown in FIG. 20.

The lysis buffer was tested to determine an optimal formula for enhancedsensitivity of a LAMP assay for SARS-CoV-2.

(a) A lysis buffer containing guanidine hydrochloride (GnHCL) (MilliporeSigma, Burlington, Mass.) was tested at various concentrations in therange of 10 mM-400 mM (1×) in combination with 1 mM, 4 mM and 8 mMTris(2-carboxyethyl)phosphine hydrochloride (TCEP)(Millipore Sigma,Burlington, Mass.) (1×) with or without 75 mM LiCl (NEB product 620151)(1×) against a SARS-CoV-2 virus titer that was varied for differentsamples containing 5,000 cps/ml, 10,000 cps/ml, 20,000 cps/ml or 40,000cps/ml (from a stock solution of 100,000 cps/ml from SeraCare, Milford,Mass.). The virus was spiked into saliva of 20 μl, 30 μl, 35 μl and 37.5μl volumes with TCEP and GnHCL. Copies of actin RNA at 100 copies/μlsaliva was used as a control. Lysis of the virus in saliva added to thesaliva lysis mix occurred at 95° C. for 5 minutes (the 10× lysis buffercontained 100 mM-4 M GnHCL, 10 mM-80 mM TCEP and 750 mM LiCl)

N2+E1 primer sets were added to a LAMP master mix (New England Biolabs,Ipswich, Mass.) containing reverse transcriptase to amplify SARS-CoV-2virus derived RNA.

Some of the results are shown in FIG. 21A and FIG. 21B (400 mM GnHCL andvarying TCEP concentration, pH and LiCl concentration for 20,000 cps/mlwhere final concentrations are given), FIG. 22 showing the effect ofincreasing the concentration of TCEP, and FIG. 23A-FIG. 23D where theLAMP reaction time was varied from 35 minutes to 60 minutes with andwithout LiCl.

The following conditions provided 100% detection from 16 samples (16/16)containing 40 virus particles/sample under the following conditions for10× lysis buffer: 4M GnHCL, 40 mM TCEP and 750 mM LiCl pH 8. 5 μl ofbuffer was combined with 45 μl of sample (35 μl saliva spiked with 10 μlof SeraCare). After a heating step at 95° C. for 5 minutes, 2 μl of thetreated saliva sample was then added to 18 μl of a LAMP master mix (10μl of 2× stock from NEB product M1800, 0.8 μl 25× primer set N2 and 0.8μl 25× primer set E1, 0.4 μl 50× dye and 6 μl water to a total of 18 μl)and incubated at 65° C. for 35 minutes. The lysis buffer was found to becompatible with colorimetric LAMP (FIG. 21A-21D to FIG. 23A-23D),fluorescent LAMP and RT-qPCR (FIG. 24). It should be noted that where alysis buffer is used that contained guanidine salt, it was not necessaryto add the guanidine salt to the master mix because of the carryover ofthis salt from the lysis buffer.

Control Primer Sequences: hActin (5′-3′)

ACTB-F3 AGTACCCCATCGAGCACG (SEQ ID NO: 96) ACTB-B3 AGCCTGGATAGCAACGTACA(SEQ ID NO: 97) ACTB-FIP GAGCCACACGCAGCTCATT GTATCACCAACTGGG ACGACA(SEQ ID NO: 98) ACTB-BIP CTGAACCCCAAGGCCAACCG GCTGGGGTGTTGAAGG TC(SEQ ID NO: 99) ACTB-LoopF TGTGGTGCCAGATTTTCTC CA (SEQ ID NO: 100)ACTB-LoopB CGAGAAGATGACCCAGATCAT GT (SEQ ID NO: 102)

Example 10: Detection of Polynucleotides in Saliva Samples Using RT-qPCR

The RT-QPCR reaction was set up as follows:

20 μl FINAL COMPONENT REACTION CONCENTRATION Luna Universal ProbeOne-Step  10 μl 1X Reaction Mix (2X) Luna WarmStart ® RT Enzyme   1 μl1X Mix (20X) Forward primer (10 μM) 0.8 μl 0.4 μM Reverse primer (10 μM)0.8 μl 0.4 μM Probe (10 μM) 0.4 μl 0.2 μM Template RNA   2 μlNuclease-free Water   5 μl

The thermal cycler was set up as follows:

CYCLE STEP TEMPERATURE TIME CYCLES Reverse Transcription 55° C.* 10minutes 1 Initial Denaturation 95° C.  1 minute 1 Denaturation 95° C. 10seconds 45 Extension 60° C. CV seconds** (+plate read)

SARS-CoV-2 RNA (Twist Biosciences, San Francisco, Calif.) and virus(SeraCare, Milford, Mass.) were spiked separately into different tubesof 2× saliva lysis buffer. The virus was previously spiked into saliva.A positive control contained purified RNA and a negative controlcontained water only. The results are consisted with those reported forRT QPCR from saliva or nasopharyngeal swabs where detection limits wereascertained from 10 copies/sample (5000 cps/ml) up to 100,000 cps/ml.

Example 11: Lysis Buffer has Minimal or No Adverse Effects on RT-qPCR oron LAMP

The lysis buffer as a whole had no adverse effects on the sensitivity ofRT-QPCR or pH-dependent colorimetric LAMP. The results are shown in FIG.24.

Example 12: Automation of a Workflow to Achieve a Throughput of 100,000Reactions in about 20 Hours

A workflow that is capable of delivering high volume throughput isillustrated in FIG. 28A-FIG. 28F and may include the followinginstruments: RNA collection tubes from Ora (Ottawa, Canada) (ORE-100),96-384 tube to plate sample transfer (Bravo with 96 or 384 ST Head fromAgilent, Santa Clara, Calif.), 384 well plate consumables (Corning,N.Y.), 384 well filling LAMP master mix into detection plates (BioTek,Winooski, Vt.), heat sealing of plates (Thermo Fisher, Waltham, Mass.),Automated 65° C. timed incubation (StoreX-Liconic Instruments) or Intekconveyer (Intek WA), Endpoint fluorescence (BioTek, Winooski, Vt.) orSpectraMax. Although any of these instruments can be switched out forother comparable devices, the workflow illustrates the suitability ofcolorimetric LAMP for high throughput workflows that are relativelysimple, cost effective, efficient, and sensitive.

Example 13: A Surfactant Containing Buffer for Use in a SensitiveNucleic Acid Assay for Detecting SARS-CoV-2 in Saliva

A buffer was formulated at a concentration that was a suitable multipleof the final concentration for adding to a volume of sample. Althoughthe buffer may be formulated in high concentrations such as 10× or 5×, aconcentration of 2× sample buffer was used here as this offered ease ofmixing with the relatively viscous saliva sample.

The NEB sample 2× buffer (2× contains 8 mM TCEP/NaOH (pH 8.2) 800 mMGuHCl, 150 mM LiCl, 0.2 mM EDTA, 0.4% PF68) was found to be stable overmonths at 2× concentration. The final concentration of the sample buffer(NEB buffer) combined with saliva was: 4 mM TCEP (pH 8.2) 400 mM GuHCl,75 mM LiCl, 0.1 mM EDTA, 0.2% PF68.

The CEPKO buffer (1× contains 2.5 mM TCEP, 11 mM NaOH, 1 mM EDTA) wasfound to be stable at a 100× concentration but was not stable at 2× or5× concentration and 100× Cepko buffer was made freshly for eachexperiment.

Experimentation showed that a reducing agent in the buffer (TCEP) couldbe used within the range of 1 mM-8 mM and pH adjusted to pH 8 with NaOH.Guanidinium hydrochloride was preferably included in the sample bufferfor testing using pH colorimetric LAMP. The surfactant used in thisexample was the poloxamer PF68. Although used here at 0.2%, theconcentration can be anywhere in the range of 0.1%-1%. In the aboveformulation, EDTA was used at 0.1 mM but the concentration of EDTA canbe in the range of 0.1%-1%.

The effect of Saliva on the sensitivity of LAMP In this example, heatinactivated SARS-CoV-2 (3.75×10⁸/cps/ml) or gamma irradiated SARS-CoV-2(1.75×10⁹ cps/ml) supplied by BEI Resources or from ATCC was seriallydiluted to determine whether the presence of saliva could adverselyaffect the sensitivity of the assay. The assay was carried out asfollows:

16 μL virus either in saliva or combined with 4 μL 5×NEB sample bufferwas serially diluted 1:10 in 1× buffer. All samples were heated at 95°C. for 5 minutes and 2 μL was added to each LAMP reaction using E1 andN2 sets of primers as provided in Example 3 and WarmStart® ColorimetricLAMP 2× Master Mix with UDG (M1804 from New England Biolabs) orWarmStart® Colorimetric LAMP 2× Master Mix (DNA & RNA) (M1800 from NewEngland Biolabs).

-   -   (a) Virus was heat inactivated SARS-CoV-2 (not in saliva). 60        copies of virus could be detected in 100% of the samples and 6        copies of virus could be detected in about 85% of the samples        tested using colorimetric LAMP (observing pH dependent color        change) and also using fluorescence based LAMP (measuring Cq).    -   (b) Virus was Gamma irradiated SARS-CoV-2 (not in saliva). 28        copies of virus at 100% and 3 copies of virus could be detected        in 50% of samples.    -   (c) The sensitivity of the assay was determined as performed        in (a) and (b) but this time virus was added to saliva before        the serial dilution in the sample buffer and heat treatment        prior to performing LAMP. The sensitivity of the assay was found        to vary in the presence of saliva resulting in some inhibition        depending on the source of the saliva. However, the use of PF68        prior and during the first heating step significantly improved        the sensitivity of the assays for virus in saliva.

Example 14: Improvements in Isothermal Amplification of Nucleic Acidsfrom RNA Viruses in Saliva

We observed that RNase activity is very high in saliva and although adetergent such as Tween and Triton X is effective at solubilizing viruscapsids at room temperature and efficiently releasing RNA, thesedetergents also make the target RNA more susceptible to RNasedegradation.

We selected a different detergent, namely an alkoxylated alcohol thatdoes not appear to solubilize the virus at room temperature or rathersolubilized virions very slowly at room temperature but rather it actsat the high temperatures used to inactivate RNases so the result wasthat the yield of viral RNA was improved.

When saliva spiked with a known amount of virus was (1) treated withheat (95° C. for 5 minutes); (2) treated with buffer containing PluronicF68 (PF68); (3) treated with heat and buffer containing Pluronic F68,and tested for RNase activity, only the combination of heat and buffer(PF68) was found to sufficiently inactivate RNase activity in freshsaliva to protect the RNA from degradation.

This result is important as it means that saliva samples combined with abuffer containing an alkoxylated alcohol can provide greatersensitivity.

The following methods of collection of saliva were used:

-   -   A. Collection of saliva for storage of samples until analysis.        -   (a) A container with sample saliva was heated for 30 minutes            at 65° C. to inactivate virus. Samples can be treated            immediately or stored at 4° C. or −20° C. for several days.        -   (b) NEB buffer containing an alkoxylated alcohol e.g. PF68            was added to the sample at the start of analysis. The sample            was found to be stable at room temperature for up to 6            hours. This is helpful when large numbers of samples are            being processed so that the samples may be maintained on the            bench until ready to perform the LAMP reaction.

The samples were then heated to 95° C. for 5 minutes. This step releasesRNA from inactivated virus while the alkoxylated alcohol in the buffersubstantially reduced RNase activity. In contrast, RNase activityremained very high after the samples were heated at 95° C. for 5 minutesin the absence of buffer. This corresponded to an absence of detectionusing LAMP. If other detergents such as Triton X-100 or Tween-20 wereused in buffer, even though after the heating step, the remaining RNaseactivity requires the samples to be stored on ice to prevent the virusRNA degradation.

NEB 1× buffer formulation in the saliva sample in this example was 400mM GnHCL, 4.5 mM TCEP pH 8.2, 75 mM LiCl and 0.2% PF68. The reagentbuffer was prepared as a 2× formulation containing 800 mM GnHCL, 9 mMTCEP pH 8.2, 150 mM LiCl, and 0.4% PF68.

-   -   B. Collection of saliva in container in which the NEB buffer is        released from a capsule in the lid. No 65° C./30 minutes heating        step is required to inactivate virus. Sample was good at room        temperature for up to 6 hours.

Heat saliva sample to 95° C. for 5 minutes to release RNA from thecapsids in the saliva.

After heat treatment, the sample should be used in LAMP detected as soonas possible. If samples have to be stored, they should be stored at −20°C. or preferably −80 C. Store 1A or 1B on ice for up to a couple of daysas needed, longer at −20° C.

An aliquot of saliva sample was removed and diluted 10 fold into LAMPreaction kit (e.g. 2 μl of saliva sample into 18 μl of LAMP reactionmix).

Results: 20 cps of virus mixed with saliva could be detected 100%sensitivity for 12/12 samples tested using saliva from a single source.

Saliva samples were collected from multiple donors and spiked withattenuated RNA virus particles. 5 μL of SeraCare (mock virus with 100cps/μL) was added to 30 μL of Saliva, 5 μL of water and 10 μL of 5×NEBbuffer. Therefore, the original titer in mixed SeraCare-Saliva samplewas 14.3 copies/μL and after adding buffer and water, the titer was 10copies/μL.

NEBuffer (2×NEB inactivation buffer formulation as a reagent: 800 mMGnHCL, 9 mM TCEP pH 8.2.150 mM LiCl and 0.4% PF68 (no Proteinase Kincluded here)) was added to the saliva. Samples were stable with noobservable decrease in detectable copies of viral RNA even when storedat room temperature for at least 6 hours. Samples were then heat at 95°C. for 5 minutes and stored on ice. Aliquots were then used for LAMPdetection or RT-qPCR as described in previous examples.

PF68 improved the sensitivity of the LAMP reaction where 20 copies ofvirus mixed with saliva from different donors could be detected with100% sensitivity.

1A) Collection of saliva for storage of samples until analysis.

When ready to analyze, add buffer (should contain preferred non-ionicdetergent e.g. PF68) (Sample is good at RT for up to 6 hours) and heatto 95° C. for 5 minutes. This step releases RNA from inactivated virusand detergent in buffer with detergent substantially reduces RNaseactivity. In contrast, RNase activity remains very high after thesamples were heated at 95° C. for 5 minutes in the absence of buffer,therefore no SARS-CoV-2 signal can be detected. If other detergents suchas Triton X-100 or Tween-20 were used in buffer, even though after theheating step RNase activity was significantly reduced, the remainingRNase activity requires the samples to be stored on ice to prevent thevirus RNA degradation.

1B) Collection of saliva in container in which the buffer is releasedfrom a capsule in the lid. No 65° C./30 minutes heating step required toinactivate virus. Sample is good at RT for up to 6 hours.

Heat 95° C. for 5 minutes.

After heat treatment, sample should be used in LAMP detected as soon aspossible. If samples have to be stored, they should be stored at −20° C.or preferably −80° C. Store 1A or 1B on ice for up to a couple of daysas needed, longer at −20° C.

Take an aliquot of saliva sample and dilute 10 fold into LAMP reactionkit (e.g. 2 ul of saliva sample into 18 μl of LAMP reaction mix.

Results: 20 cps of virus mixed with saliva can be detected 100%sensitivity for 12/12 samples tested using saliva from 1 person.

Example 15: An Alternate Protocol for SARS-CoV-2 Screening

Saliva was collected in 1.5 ml barcoded tubes and heated to 65° C. for30 minutes at the collection site in a buffer that contained 0.2% PF68or Triton X-100 or no detergent. In this example, the sample was spikedwith non-infectious heat inactivated SARS-CoV-2 virus grown in cellculture (ATCC).

25 μL of saliva was mixed with 25 μL of 2× inactivation buffer (1× Cepkoinactivation buffer: 2.5 mM TCEP, 11 mM NaOH and 1 mM EDTA)—see Apr. 28,2020 (https://doi.org/10.1101/2020.04.23.20076877) or NEBuffer (1×inactivation buffer: 4 mM TCEP, 1 mM EDTA, 25 mM LiCl and 400 mM GnCl).When Triton X −100 was added to the Cepko buffer, it was found that theRNA was not preserved at room temperature.

A space change multichannel pipette was used to transfer saliva into96-well plate with buffer and mixed samples were heated at 95° C. for 5minutes. 2 μL of sample was transferred into a LAMP reaction followed bya 65° C. 30 minutes amplification and data collection by observing acolor change in a tube. The protocol is described in FIG. 30. Examplesof the results obtained are shown in FIG. 31A-31C.

Sample in buffer that did not contain detergent could be stored on iceto preserve viral RNA prior to the 95° C. heat step. If Triton X-100 wasadded to the buffer, it was found that the RNA was not preserved even atroom temperature. However, when Pf68 was added to either CepKo bufferand NEBuffer, the sensitivity of both assays were increased compared tothe use of Triton-100 despite some variability associated with salivasamples from different human subjects.

Example 16: Immobilization of Oligonucleotide Reagents to EnhanceSensitivity and/or Improve Efficiency of Workflow for Diagnosis ofSARS-CoV-2

Immobilized reagents provide an opportunity to achieve enrichment oftarget nucleic acid from a biological sample and/or reduce the number ofsteps in a workflow.

This example describes the application of bead immobilized reagents toimprove a coronavirus testing workflow that utilizes saliva as astarting material to test for the presence of SARS-CoV-2 virus. and usesLAMP to amplify cDNA derived from any SARS-CoV-2 RNA from virus presentin the saliva to determine whether an individual is infected. Humansaliva is used and an attenuated SARS-CoV-2 is spiked into the saliva atknown concentrations.

The saliva is collected in a first receiving vessel containing a bufferthat includes a surfactant/detergent. Enrichment of the SARS-CoV-2 RNAis initiated by the release of viral nucleic acid from virus followed byhybridization of the released RNA to biotinylated oligonucleotidesimmobilized on streptavidin coated magnetic beads or oligonucleotidecoated glass beads. In this test, we use a non-ionic detergent-PluronicF-68 as the surfactant in the receiving vessel. The contents of thereceiving tube is varied as described below and may additionally containone or more RNase inhibitors and a proteolytic agent.

-   -   1. Buffer contains PF-68, magnetic beads coated with a DNA        oligonucleotide primer for initiating reverse transcription plus        heat labile Proteinase K.    -   2. Buffer contains the Pf-68, magnetic beads coated with a DNA        oligonucleotide primer for initiating reverse transcription        absent heat labile Proteinase K, plus guanidinium chloride.    -   3. Buffer contains PF-68, glass (or any silica-derived) beads of        at least 1 mm diameter optionally coated with a DNA        oligonucleotide primer for initiating reverse transcription plus        heat labile Proteinase K.    -   4. Buffer contains PF-68, glass (or any silica-derived) beads of        at least 1 mm diameter optionally coated with a DNA        oligonucleotide primer for initiating reverse transcription        absent heat labile Proteinase K, plus guanidinium chloride.    -   5. Buffer contains PF-68, magnetic beads coated with an RNA or        DNA oligonucleotide primer for binding the viral RNA at a        predetermined location (e.g. EIA) plus heat labile Proteinase K.    -   6. Buffer contains PF-68, magnetic beads coated with coated with        an RNA or DNA oligonucleotide primer for binding the viral RNA        at a predetermined location (e.g. EIA) absent heat labile        Proteinase K, plus guanidinium chloride.    -   7. Buffer contains PF-68, glass (or any silica-derived) beads of        at least 1 mm diameter optionally coated with an RNA or DNA        oligonucleotide primer for binding the viral RNA at a        predetermined location (e.g. EIA) plus heat labile Proteinase K.    -   8. Buffer contains PF-68, glass beads (or any silica-derived) of        at least 1 mm diameter optionally coated with an RNA or DNA        oligonucleotide primer for binding the viral RNA at a        predetermined location (e.g. EIA) absent heat labile Proteinase        K, plus guanidinium chloride.

Workflow Details

Following the binding step, the magnetic beads containing the hybridizedviral genome are separated from the buffer by means of a magnet, wherethe buffer containing unwanted biological material is discarded and thebeads suspended in a buffer containing reagents for reversetranscription and amplification by LAMP (see RT-LAMP from New EnglandBiolabs, Ipswich, Mass.).

For the glass beads containing the hybridized viral genome or target(s),a filter with a pore size less than the diameter of the beads is placedin a sleeve within a second receiving vessel (see U.S. patentapplication Ser. No. 16/547,844 and New England Biolabs, Ipswich, Mass.)In this case, the receiving vessel has an exit that has an open and shutposition. In the open position, the buffer containing the unwantedbiological material is drained away and the sleeve placed into a secondtube containing a buffer suitable for amplification (e.g., RT-LAMP,RT-qPCR, etc.).

Hypothesized Results

Where magnetic beads were used, enrichment of viral RNA only wasachieved and hence cDNA was also specific for the target viral RNA. Incontrast, use of glass beads would result in adsorption of somenon-target viral RNA and hence cDNA would include non-viral cDNA.Nonetheless this contamination would have negligible impact since theplurality of sets of probes for LAMP are highly specific for the targetviral genome.

If a person is infected with SARS-CoV-2, the saliva is expected tocontain between 10,000-20,000 viral particles. For a positive result tobe obtained using LAMP, it is desirable to be able to detect thepresence of at least 25 viral genomes. The immobilization ofoligonucleotide probes and enrichment of target nucleic acids fromsaliva is expected to deliver this degree of sensitivity even ifhybridization of target viral RNA to immobilized probes is not 100%efficient. In fact, even if this were less than 5% efficient, thesensitivity and reproducibility of the LAMP based test would be expectedto increase besides any loss of efficiency in handling large numbers ofsamples or significant increase in workflow complexity.

Example 17: Detection of Influenza and SARS-CoV-2 in a MultiplexReaction

Multiplex Reaction-Selection of Primer Sets

For Influenza detection, a variety of LAMP primer sets reported byothers (Parida et al. J Mol Diagn 13(1), 100-107 (2011), Mahony et al. JClin Virol 58(1), 127-131 (2013), Ahn et al. BMC Infect Dis 19(1), 676(2019) and Takayama et al. J Virol Methods 267 53-58 (2019)) weresynthesized, tested and compared with respect to compatibility andutility in DARQ LAMP. These primer sets were also evaluated for speed,sensitivity, and ability to multiplex with the SARS-CoV-2 sets. Fromthis comparison we selected the IAV and IBV primer sets from Takayama etal. as they were found to be the most sensitive primer sets for DARQLAMP. The multiplex LAMP tested contained the IAV and IAB primer setsfor Influenza, the E1 primer set for SARS-CoV-2 and ACTB for an internalcontrol.

Materials and Methods

DARQ LAMP primer sets (F3, B3, FIP, BIP, LF and LB) plus a duplex oligoconsisting of the FIP modified at its 5′-end with a dark quencher(Q-FIP) annealed to a complementary F1c oligo with 3′ fluorophore (Fd)(Table 1) were selected for target RNAs. The E1 LAMP primers targetingSARS-CoV-2 sequence (GenBank accession number MN908947) and IAV and IBVprimer sets (Takayama, et al). were synthesized at Integrated DNATechnologies (Coralville, Iowa) with standard desalting for conventionalLAMP primers and HPLC purification for QFIP and Fd oligos. SyntheticSARS-CoV-2 RNA containing equal ratio of the viral genome regions waspurchased from Twist Bioscience, Califorina (Twist Synthetic SARS-CoV-2RNA Control 2 #102024, MN908947.3). RNAs for Influenza A (H1N1)(VR-1737D, Strain A/Virginia/ATCC/2009), A (H3N2) (VR-1811D, StrainA/Virginia/ATCC6/2012) and B (VR-1885DQ, Strain B/Wisconsin/1/2010BX-41A) were purchased from ATCC. Viral RNA was diluted to lowerconcentrations in 10 ng/μl Jurkat total RNA (BioChain) based onquantification provided by the manufacturers. For the 24 repeatreactions, the amount of RNA used was 50 copies of SARS-CoV-2 RNA, 1 ulof 1:1000 diluted Influenza A RNA and approximately 21 copies forInfluenza B RNA. This amount of viral RNAs was sufficient for more thanhalf but not all the 24 repeats to show positive amplification thusallowing detection of sensitivity change under different conditions.

Primer sets are shown in Table 3:

TABLE 3 Primer set Sequence E1[11] E1-F3 TGAGTACGAACTTATGTACTCAT(SEQ ID NO: 70) E1-B3 TTCAGATTTTTAACACGAGAGT (SEQ ID NO: 71) E1-FIPACCACGAAAGCAAGAAAAAGAAG TTCGTTTCGGAAGAGACAG (SEQ ID NO: 72 E1-BIPTTGCTAGTTACACTAGCCATCCT TAGGTTTTACAAGACTCACGT (SEQ ID NO: 73) E1-LFCGCTATTAACTATTAACG (SEQ ID NO: 74) E1-LB GCGCTTCGATTGTGTGCGT(SEQ ID NO: 75) E1-QFIP /5IABkFQ/ACCACGAAAGCAAG AAAAAGAAGTTCGTTTCGGAAGAGACAG (SEQ ID NO: 76) E1-FD ACTTCTTTTTCTTGCTTTCGTGG T/3Joe_N/(SEQ ID NO: 77) IAV IAV-F3-1 GACTTGAAGATGTCTTTGC (SEQ ID NO: 78)IAV-F3-2 GACTGGAAAGTGTCTTTGC (SEQ ID NO: 79) IAV-B3-1TRTTATTTGGGTCTCCATT (SEQ ID NO: 80) IAV-B3-2 TRTTGTTTGGGTCCCCATT(SEQ ID NO: 81) IAV-FIP TTAGTCAGAGGTGACARRATTGC AGATCTTGAGGCTCTC(SEQ ID NO: 82) IAV-BIP TTGTKTTCACGCTCACCGTGTTT GGACAAAGCGTCTACG(SEQ ID NO: 83) IAV-LF GTCTTGTCTTTAGCCA (SEQ ID NO: 84) IAV-LBCMAGTGAGCGAGGACTG (SEQ ID NO: 85) IAV-QFIP /5IAbRQ/TTAGTCAGAGGTGACARRATTGCAGATCTTGAGGCTCT C (SEQ ID NO: 86) IAV-Fd CAATYYTGTCACCTCTGACTAA/3Cy5Sp/ (SEQ ID NO: 87) IBV IBV-F3 GCAACCAATGCCACCATA (SEQ ID NO: 88)IBV-B3 TTCTCTCTTCAAGRGACATC (SEQ ID NO: 89) IBV-FIPTAGTCAAGGGCYCTTTGCCACTT TGAAGCAGGAATTCTGGA (SEQ ID NO: 90) IBV-BIPCAAGACCGCCTAAACAGACTAAA CTTTTACTTTCAGGCTCACTT (SEQ ID NO: 91) IBV-LFTGAAAGYCTTTCATAGCAC (SEQ ID NO: 92) IBV-LB CAAGAATAAAGACTCACAAC(SEQ ID NO: 93) IBV-QFIP /5IABkFQ/TAGTCAAGGGCYC TTTGCCACTTTGAAGCAGGAATTCTGGA (SEQ ID NO: 94) IBV-Fd TGGCAAAGRGCCCTTGACTA/ 36-FAM/(SEQ ID NO: 95) ACTB ACTB-F3 AGTACCCCATCGAGCACG (SEQ ID NO: 96) ACTB-B3AGCCTGGATAGCAACGTACA (SEQ ID NO: 97) ACTB-FIP GAGCCACACGCAGCTCATTGTATCACCAACTGGGACGACA (SEQ ID NO: 98) ACTB-BIP CTGAACCCCAAGGCCAACCGGCTGGGGTGTTGAAGGTC (SEQ ID NO: 99) ACTB-LF TGTGGTGCCAGATTTTCTCCA(SEQ ID NO: 100) ACTB-LB CGAGAAGATGACCCAGATCAT GT (SEQ ID NO: 102)ACTB-QFIP /5IAbRQ/GAGCCACACGCAG CTCATTGTATCACCAACTGGG ACGACA(SEQ ID NO: 103) ACTB-Fd TACAATGAGCTGCGTGTGGCT C/3Rox_N/(SEQ ID NO: 104)

All Influenza primers were initially screened for performance usingWarmStart® Colorimetric LAMP 2× Master Mix (DNA & RNA) (New EnglandBiolabs, M1800) and with WarmStart LAMP Kit (DNA & RNA) (New EnglandBiolabs, E1700) supplemented with 1 μM SYTO®-9 double-stranded DNAbinding dye (ThermoFisher, S34854). DARQ LAMP reactions contained1×E1700 (New England Biolabs), with an additional 0.32 U/μL Bst 2.0WarmStart DNA Polymerase (New England Biolabs, M0538), 40 mM guanidinechloride (Sigma, RDD001) and standard concentrations of LAMP primers(0.2 μM F3, 0.2 μM B3, 1.6 μM FIP, 1.6 μM BIP, 0.4 μM LoopF, 0.4 μM LoopB) supplemented with DARQ FIP duplex (0.22 μM QFIP: 0.18 μM Fd,pre-annealed as 55 μM QFIP: 45 μM Fd by heating to 95° C. and slowlycooling to room temperature). The concentration for ACTB primers wasreduced to 0.25× the standard concentrations for LAMP primers (0.05 μMF3, 0.05 μM B3, 0.4 μM FIP, 0.4 μM BIP, 0.1 μM LoopF, 0.1 μM Loop B)with DARQ FIP duplex added as 0.066 μM QFIP and 0.054 μM Fd. Thereactions were incubated at 60° C. in half-skirted plates (BioRad,HSP9601) on a real-time qPCR machine (Bio-Rad CFX96). Real time LAMPsignal was acquired every 15 seconds for 108 cycles (total incubationtime ˜40 minutes for single channel or 49 minutes for 4-channelacquisition). Completed LAMP reactions were then scanned on BioTekSynergy NEO 2 microplate reader for fluorescence signal for 5-FAM(Excitation, 484/20, Emission 530/25, Signal Gain 75), HEX (524/20,565/20, 75), 5-ROX (569/20, 615/25, 85) and Cy5 (640/20, 682/20, 75).The threshold for the positive signal was set as above the sum ofaverage raw fluorescence units (RFU) of 8 NTC reactions plus 10× oftheir standard deviation.

The results showed that the amplification speed (determined from Cq) anddetection sensitivity (plots of RFU from endpoint scanning) was similarfor conventional LAMP monitored using Syto-9 and for DARQ-LAMP. 24repeats with 50 copies of SARS-CoV-2 RNA or 8 repeats of NTC were testedusing E1 or E1 and ACTB. Sensitivity was also tested for SARS-CoV-2 inthe presence of (E1+ACTB) (E1+ACTB+IAV) and (E1+ACTB+IAV+IBV) primersets by DARQ LAMP (see for example FIG. 31A-31C).

Example 18: SARS-Cov-2 RT LAMP Assay Validation for Saliva Samples

The conditions for performing a LAMP reaction on saliva samples todetect SARS-Cov-2 were stringently tested to provide a method that wasaccurate and reproducible not just for a single operator or technicianbut for multiple operators and technicians. The results below show thatthis was achieved successfully. Moreover, this approach has been testedsuccessfully on another RNA virus, namely Eastern equine encephalitisusing crushed mosquitos that contain virus infected blood from mammalianhosts using a single set of LAMP primers. RT-LAMP detects the geneticmaterial present in the SARS-CoV-2 virus. The RNA genome is firstreverse transcribed to create a complementary DNA (cDNA) strand.Specific cDNA primers then amplify conserved SARS-CoV-2 regions withintwo targets, the N and E genes. Amplification is detected by increasedfluorescence as the included dye intercalates in the newly created cDNA.In addition, a pH indicator dye signals a change in pH resulting fromthe release of H+ as nucleotide triphosphates are incorporated into thecDNA amplification product. In the absence of the target N and E regionsno cDNA amplification occurs, and hence no fluorescent signal increaseor pH indicator change is noted. An analogous reaction targeting humanactin RNA is run separately on each sample to ensure the sample isdevoid of reaction inhibitors and contains sufficient material forbaseline detection.

Cq refers to the cycle number in which the fluorescence signal passes athreshold. If no signal above threshold can be detected, Cq is reportedas N/A (not available). The more target RNA present, the smaller the Cqvalue obtained. In the current protocol, the fluorescence signal is readat 15 second intervals throughout the reaction period of 36 minutes.

Color change indicates the presence of a target nucleic acid. Color toneof LAMP without target is pink and with detectable target is yellow.Amplification is determined visually and recorded using a scanner whereyellow indicates virus is present and pink indicates that the virus isabsent. Sometimes the color is orange if the reaction contains aninhibitory substance or a very low copy number of virus.

A. Reagents/Solutions

Primer sets for both SARS-CoV-2 and actin RNA detection, TCEP,Hydrochloride solid (Millipore Sigma), Sodium hydroxide (NaOH) solution,EDTA solution, Pluronic F-68 (PF68) Non-ionic Surfactant (ThermoFisherScientific), Nuclease-free water (New England Biolabs)

B. Controls: Inactivated SARS-CoV-2 Positive Control (BEI Resources),Human Total RNA Positive Control (ThermoFisher)

C. Equipment: Bio-Rad CFX96 qPCR instrument, Bio-Rad Thermal Cycler.Eppendorf Xplorer® plus, single-channel, 5-100 μL electronic pipette,8-channel, 15-300 μL electronic pipette, 12-channel, 5-100 μL electronicpipette.

D. Buffers

1. 100× Stock Buffer (10 mL)

TABLE 4 Stock Final Concentration Component Concentration (100x) 10 mLTCEP 0.5M 0.25M  5 mL NaOH  10M  1.1M 1.1 mL  EDTA 0.5M  0.1M  2 mLNuclease 1.1 mL  free H₂O Total 10 mL

2. 2× Saliva Lysis Buffer (SLB) Buffer (100 mL)

TABLE 5 Stock Component Concentration 100 mL 2X Stock buffer 100X  2 mL10% Pluronic F-68  50X  4 mL Nuclease free H₂O 94 mL Total 100 mL 

Method of Analysis of the Saliva Sample

An inactivated SARS-CoV-2 Positive Control was obtained from BEIresources (Manassas, Va.) Pooled human saliva samples negative forSARS-CoV-2 (from NEB research study) were also tested. Human total RNAwas monitored with a sample extraction and inhibition control usingdetection of actin mRNA where actin mRNA was obtained from ThermoFisher(MA). SARS-CoV-2 positive and negative clinical saliva samples provideby Mirimus Clinical Labs, a CLIA-certified facility where SARS-CoV-2sample status was classified by Mirimus following RNA extraction usingThermoFisher TaqPath. 30 positive and 30 negative samples were analyzedin this example. The primers used in LAMP targeting gene N, Orflab, andS genes.

Saliva samples were heated in 1.5 mL labelled tubes at 65° C. for 30 minto inactivate any SARS-CoV2, and subsequently stored on ice or in therefrigerator for up to 72 hours. 154 of individual heat-inactivated testsamples were transferred into 94 of the individual wells of a 96 wellSLB plate. Up to 94 test samples were added into each 96 well SLB plate.One well was reserved for the negative control (NC) and one for thepositive control (PC). See an example plate in FIG. 33.

-   -   1. Controls        -   a. 15 μl of H₂O was added to each of the two plates, in            negative control well        -   b. 15 μL of H₂O containing 50 copies of heat inactivated            virus was dispensed into the positive control well in the            SARS-CoV-2 test plate.    -   2. Sample plates were heated for 95° C. for 5 minutes then held        at 4° C. The samples were stored at 0-4° C. for up to 24 hours        prior to performing the LAMP detection reaction.    -   A. Preparation of SARS-CoV-2 LAMP reaction plate and actin LAMP        reaction plate in AirClean PCR Workstation        -   1. A 2 mL Eppendorf tube was labelled for SARS-CoV-2            reaction mix and another for human actin reaction mix and            both were placed on ice.        -   2. The following SARS-CoV-2 RT-LAMP reaction mix was            prepared for one 96-well plate:

TABLE 6 Volume/100 Component Volume/reaction reactions 2x Master Mix,(NEB: E2019) 10 μl 1000 μl  10X SARS-CoV-2 primer mix  2 μl 200 μl (NEB:S1883*) 10X GuHCl solution  2 μl 200 μl 50X Syto 9 dye 0.4 μl   40 μlNuclease free H₂O 3.6 μl  360 μl Total 18 μl

indicates data missing or illegible when filed

-   -   3. Prepare actin RT-LAMP reaction mix for one 96-well plate:

TABLE 7 Volume/100 Component Volume/reaction reactions 2x Master Mix,(NEB: E2019) 10 μl 1000 μl 10X actin primer mix  2 μl  200 μl (NEB:S0164*) 10X GuHCl solution  2 μl  200 μl 50X Syto 9 dye 0.4 μl   40 μlNuclease free H₂O 3.6 μl   360 μl Total 18 μl 1800 μl *NEB: E2019RT-LAMP kit contains two 10X Primer mixes. The SARS-CoV-2 LAMP reactionuses primer mix S1883, and the actin LAMP reaction uses primer mixS0164.

18 μL SARS-CoV-2 LAMP reaction mix was distributed to each well of theSARS-CoV-2 reaction plate. 18 μL actin LAMP reaction mix was distributedto each well of the actin reaction plate.

An inactivated SARS-CoV-2 Positive Control was obtained from BEIresources (Manassas, Va.) Pooled human saliva samples negative forSARS-CoV-2 (from NEB research study) were also tested. Human total RNAwas monitored with a sample extraction and inhibition control usingdetection of actin mRNA where actin mRNA was obtained from ThermoFisher(MA). SARS-CoV-2 positive and negative clinical saliva samples provideby Mirimus Clinical Labs, a CLIA-certified facility where SARS-CoV-2sample status was classified by Mirimus following RNA extraction usingThermoFisher TaqPath. 30 positive and 30 negative samples were analyzedin this example. The primers used in LAMP targeting gene N, Orflab, andS genes.

2 μL prepared sample and the actin control were transferred from the SLBplate to the SARS-CoV-2 LAMP reaction plate containing 18 ul of LAMPreaction mi. 2 μL of 1 ng/μL human RNA was transferred into the positivecontrol well. The thermal cycler was set to 97 cycles, each at 65° C.for 15 seconds (total time approximately 36 minutes), followed by a holdat 10° C. and fluorescence recorded during the 97 cycles. A‘post-amplification’ image of the plate was generated for visualconfirmation of results.

Using the method described above, the saliva assay was tested usingnegative saliva samples spiked with inactivated SARS-CoV-2 (contingentsamples) and clinical samples. No RNA purification was performed.

10 different negative saliva were pooled.

320 copies/uL inactivated virus (BEI) were added to negative pooledsaliva.

This spiked saliva was then diluted by a 1:2 serial dilution: 7 fold) innegative saliva. Triplicate saliva samples were prepared for eachdilution. 15 uL of saliva was combined with 15 uL of 2× sample bufferand incubated at 95° C. for 5 minutes. 2 uL of sample was added to 20 uLof a LAMP reaction mix for triplicate LAMP reactions at each dilution.Cq values were obtained. The LOD of SARS-Cov-2 detection was 40copies/ul.

Every positive SARS-CoV-2 saliva sample was retested in triplicate. Ifrepeat testing showed more than 2 positive SARS-CoV-2 results, a“Positive” determination was recorded. Otherwise, the test was deemedinconclusive. For initially inconclusive saliva samples, the assay wasrepeated using original saliva sample with triplicate LAMP reactions toconfirm the result.

Results Clinical Sample Validation

TABLE 8 Mirimus Mirimus Li_2/24/2021 Zhang_3/4/2021 Li_3/5/2021 Plate IDpositive negative Covid Actin Covid Actin Covid Actin F7 1 27.51 28.0725.44 29.42 25.62 29.39 F4 2 26.6 25.95 24.28 28.47 25.39 27.16 E2 327.39 28.05 24.92 30.86 25.51 29.6 D8 4 27.33 30.14 23.32 30.9 25.3129.76 B5 5 27.36 26.15 24.53 33.74 25.35 31.45 F1 6 26.49 26.24 26.1828.24 25.23 28.02 E3 7 26.24 26.54 24.92 32.42 25.21 27.9 D1 8 25.829.33 25.21 28.48 25.62 29.52 E5 9 26.61 26.16 24.25 29.79 25.74 28.83F3 10 26.9 30.44 25.85 30.37 24.57 29.13 E4 11 25.06 26.5 25.28 47.6425.24 31.4 H7 12 26.58 26.3 24.02 28.82 26.07 29.8 C8 13 27.17 26.9425.57 33.12 25.22 28.53 A7 14 26.39 34.69 26.21 30.54 25.31 32.15 A3 1526.72 25.6 24.83 33.87 27.19 29.19 H3 16 26.8 29.57 26.47 32.19 24.5129.1 C4 17 36.36 29.45 29.51 39.22 28.98 33.44 A4 20 40.18 63.07 29.6777.04 29.06 30.54 C3 21 32.64 28.54 27.21 38.6 N/A 30.25 B7 22 29.5728.77 26.74 31.22 31.02 29.88 H2 23 36.87 28.73 25.72 43.93 29.33 30.96D6 24 31.05 32.76 33.02 56.05 28.63 30.3 F6 25 33.44 28.04 27.3 34.631.34 31.68 A8 27 35.55 27.02 27.2 32.27 28.98 31.65 B4 28 84.32 27.8490.39 36.61 29.15 28.37 F2 29 31.6 26.83 27.82 30.18 27.67 29.18 C6 3030.87 25.35 30.17 29.76 62.49 28.02 B6 33 29.83 26.9 28.72 27.68 28.5829 H5 37 31.13 27.52 62.39 36.12 32.04 28.38 G1 39 30.48 25.88 28.4529.86 27.94 28.91 H4 1 N/A 30.57 N/A 34.07 N/A 33.17 D7 2 N/A 28.85 N/A29.15 N/A 30.55 D2 3 N/A 33.15 N/A 33.99 N/A 34.53 E6 5 N/A 29.34 N/A31.18 N/A 30.19 B1 6 N/A 27.26 N/A 31.34 N/A 29.49 G3 7 N/A 31.03 N/A33.54 N/A 34.47 G7 8 N/A 28.77 N/A 33.03 N/A 30.21 E1 9 N/A 28.55 88.7931.07 86.9 29.6 D3 10 N/A 34.38 N/A 35.32 N/A 37.07 A5 11 N/A 27.79 N/A30.76 N/A 29.34 G2 12 N/A 30.74 N/A 33.17 N/A 30.47 E7 13 N/A 36.25 N/A36.37 N/A 38.04 C2 14 N/A 29.75 N/A 30.95 N/A 32.25 C5 15 N/A 29.1396.05 29.59 95.09 29.66 G4 16 N/A 31.07 N/A 32.52 N/A 32.57 D5 17 N/A27.87 N/A 30.77 N/A 29.91 D4 18 N/A 29 N/A 29.6 N/A 29.18 C7 19 N/A28.59 N/A 31.17 86.94 32.64 B3 20 N/A 29.16 N/A 30.5 N/A 30.49 A1 21 N/A28.53 N/A 33.76 96.03 31.5 A2 22 N/A 34.17 N/A 34.19 N/A 37.21 G6 23 N/A28.71 N/A 30.67 N/A 31.23 H6 24 N/A 27.15 N/A 29.76 N/A 30.34 H1 25 N/A30.1 81.5 32.02 N/A 35.03 F5 26 N/A 29.15 N/A 31.28 N/A 31.3 B2 27 N/A29.39 N/A 32.34 N/A 32.23 C1 28 N/A 29.29 N/A 30.94 82.82 30.43 A6 29N/A 28.1 81.26 30.94 N/A 30.66 G5 30 N/A 26.19 N/A 28.66 N/A 27.68 B8 31N/A 32 N/A 33.34 N/A 33.57 E8 H2O H2O N/A N/A N/A 91.06 N/A 83.94 F8 H2OH2O N/A N/A N/A 90.91 N/A 83.98 G8 Virus hRNA 24.54 20.92 23.3 20.8823.47 22.42 H8 Virus hRNA 25.08 20.62 23.89 21.02 24.03 22.39 total59/60 60/60 59/60 59/60 59/60 60/60 % 98% 100% 98% 98% 98% 100%

-   -   Cq values for the individual assays are given above. Samples        were anonymized on the plate but sorted for the table.    -   H₂O indicates a water negative control.    -   Virus indicates 50 copies of SARS-CoV-2 positive control.    -   hRNA indicates 2 ng human total RNA positive control for actin        mRNA.    -   Orange box indicates sample not meeting Mirimus assay        expectation.

The following parameters were provided.

1. Accuracy

A. Concordance with expected positive results

-   -   i. Contrived samples (virus-negative saliva spiked with known        amounts of virus)        -   1. All 96 samples with at least 40 copies/assay were            correctly identified using the assay.        -   2. All 30 samples lacking added virus were correctly            identified as negative and 30 Mirimus positive samples were            identified as positive.    -   ii. Clinical samples        -   1. Each run showed either 59/60 or 60/60 concordance with            Mirimus results        -   2. Minimally 98% accuracy for one operator and for multiple            operators at least 97% accuracy (58/60 samples)

B. Reproducibility of the assay

-   -   i. Contrived samples        -   1. All 20 virus-containing samples, each in triplicate, were            found to be positive (60 samples)        -   2. All 6 saliva samples without virus were found to be            negative    -   ii Clinical samples        -   1. In the hands of two different technicians on three            separate days at least 59 Of 60 samples matched the assay            results provided by Mirimus (98%)            -   2. Pairwise comparison of individual runs showed                agreement between runs. Testing results were consistent                (within 95%) on two separate days with two independent                technicians, using identical samples                3. Sensitivity: (True positives)/(True positives+False                negatives)

A. Determination of Limit of Detection (LOD) using contrived samples

Samples with Cq values below 70 were scored as positive for SARS-CoV-2as per assay protocol. All nine samples with at least 40 viral copies/piwere found to be positive by the assay criteria. Samples with 20 or 10viral copies/pi were found to be positive in 8/9 or 7/9 samples,respectively. No samples in the negative controls were found to bepositive. LOD was set at 40 viral copies/pi, with reliability droppingoff below that level. The following LOD values were obtained: Fromcontrived samples, 40 copies/pi: 9/(9+0)=100%; 20 copies/pi:8/(8+1)=89%; 10 copies/pi: 7/(7+2)=78%. From Clinical samples, Feb. 24,2021: 29/(29+1)=97%, 3/4/21: 29/(29+1)=97% and Mar. 5, 2021:29/(29+1)=97%

4. Specificity: (True negatives)/(True negatives+False positives)

A. from LOD determinations

-   -   i. At 40 copies/pi: 9/(9+0)=100%    -   ii. At 20 copies/pi: 9/(9+0)=1 00%    -   iii. At 10 copies/pi: 9/(9+0)=100%

B. From clinical samples

-   -   i. Feb. 24, 21 30/(30+0)=100%    -   ii. Mar. 4, 2021 30/(30+0)=100%    -   iii. Mar. 5, 2021 30/(30+0)=100%        5. Reportable values

A. Values reported as detected or undetected.

B. Reproducibility suffered at virus concentrations less than 40copies/pi.

C. Detection occurred at all tested virus concentrations above 40copies/μL.

6. Reference range:

As can be seen in the accompanying figure, the anticipated viral load insaliva ranged between 104-1010 copies/mL in patients, with decreasingviral loads as infection progresses. The LOD for this method was 40copies/pi, or 4×104 copies/mL, covering the vast majority of expectedinfection viral titers, particularly in the early stages of infection.

Example 19: Universal Assay for a Pathogen and its Known and UnknownVariants

In order to provide solutions for distinguishing sequence variantsrather than just tolerating them, we designed a variant-specific LAMPdetection method based on molecular beacons targeting a 9-base deletionin Orf1a that is more widely observed (SGF deletion), enabling thedetection of multiple variants of concern including B.1.1.7, B.1.351,and P.1.

The phrase “Target nucleic acids with (i) a known sequence or (ii) aknown sequence with a plurality of undefined mutations therein” refersto variants of a target nucleic acid in a cell or virus that have“undefined mutations”. Undefined mutations may arise in a cell or viruspathogen to evade host immune mechanisms or medications or for otherreasons. The mutations that arise may result in changed propertiesrelating to virulence and pathogenicity usually by increasing one orboth. Often these variants coexist with other mutants or variants. It isbeneficial to an animal population such as humans if the appearance of anew variant is rapidly detected before extensive spreading.

PCR is an established, sensitive method of detecting specific targetnucleic acids. that require two primers only and a thermocyclingprotocol. If novel mutations arise in a target nucleic particularly atthe 3′ end of the primer binding site, PCR will take longer to amplifythe nucleic acid to the point that a positive sample will register asnegative. A negative PCR result is one that does not yield a positivesignal after a predetermined number of thermocycles. This can havenegative repercussions for detecting infected individuals in apopulation in which multiple variants coexist where an infected personmay receive a negative result on the basis of less than ideal PCRprimers. Efficiency of primer annealing is therefore a factor inconsidering sensitivity of a PCR reaction.

PCR differs from LAMP amplification in ways that include the nature ofthe amplification itself. Without being limited by theory it is proposedhere that the inefficiencies in primer hybridization for PCR areamplified by the protocol compared with LAMP. In PCR, amplification is aseries of discrete events in which primers are required to bind to eachmolecule in the population and then to bind to each of the amplicons inevery cycle. Therefore any inefficiency in binding due to mutations isitself amplified. In contrast, LAMP uses 4, 5 or 6 primers which createintermediate species that are then amplified from multiple primerbinding events and locations, These multiple binding and amplificationsites reduce the impact of primer binding efficiencies.

The use of the term “universal primer set” is intended to refer to aprimer set used in LAMP to detect a specific gene or specific sequencein a target nucleic acid or the entire nucleic acid. The specific gene,sequence or the entire target nucleic acid may contain mutationsincluding one or more, or a plurality of previously unknown mutations.The presence of mutations in the target nucleic acid is referred to hereas target nucleic acid variants. These mutations may be additions,deletions or point mutations that occur at primer binding sites forprimers used in nucleic acid amplification assays to determine thepresence of a pathogenic cell or virus. The universal primer set is sonamed because the primers can initiate amplification reactions to detecta specific target with a similar sensitivity and specificity regardlessof known or unknown mutations in the primer binding sites of thattarget. However, it is expected that the deletions or additions will notexceed 10 nucleotides or more particularly will not exceed 9 nucleotidesfor example, will not exceed 8 nucleotides for example, will not exceed6 nucleotides within primer binding regions. In some embodiments, it wasfound that deletions of 9 nucleotides and 6 nucleotides in the FIP andBIP primer binding sites could be detected by LAMP using the universalLAMP primer set although it was noted that a deletion of 9 nucleotidescould reduce sensitivity of a diagnostic test. The universal primer setis also able to detect a mixture of nucleic acids where some nucleicacids may have a particular target sequence and others are variantsthereof. The universal primer set may include 4, 5 or 6 primers. In somecircumstances, a plurality of universal primer sets will be used todetect a target nucleic acid for amplifying different regions of thetarget nucleic acid.

LAMP is characterized by the use of 4 different primers specificallydesigned to recognize 6 distinct regions of the target gene. The fourprimers include 2 inner primers (Forward inner primer (FIP) and Backwardinner primer) and 2 outer primers (Forward outer primer (F3) andBackward outer primer (B3). A universal primer set may also include 2additional primers-Loop primers (Loop forward (LF) and Loop backward(LB) primers. Although the loop primers are optional, they can increasesensitivity and specificity of the amplification reaction.

The term “reaction positive period of time” refers to the reaction timefor amplification prior to an established cut-off time in which a sampleis determined to be positive. The cut-off time for amplification tooccur has been established by diagnostic laboratories to determine if asample is positive or negative.

Materials and Methods

Single point mutation LAMP primers: Three previously described LAMPprimer sets for SARS-CoV-2 were chosen to profile mutational positioneffects: As1e [20], E1, and N2 [4] (Table 9). A point mutation thatappeared within GISAID sequences as monitored by NEB Primer Monitor(primer-montor.neb.com) was introduced at every base position for eachof the primers changing: C→T, T→C, G→T, or A→C. The resulting 572primers were synthesized in 96-well plates at 10× concentration (2 μMF3, B3; 16 μM FIP, BIP; 4 μM LoopF, LoopB) by Integrated DNATechnologies (IDT) and spot-checked for concentration using 90 of theprovided oligos.

TABLE 9 LAMP Primers* Assay Primer Sequence SFG F3 GCTTTTGCAATGATGTTTGTC(SEQ ID NO: 1105) B3 AGTGTCCACACTCTCCTAG (SEQ ID NO: 106) FIPCCAACTAGCAGGCATATAGAC CATACATTTCTCTGTTTGTTT TTGTTACC (SEQ ID NO: 107)BIP ATGACATGGTTGGATATGGTT GGTTCTTGCTGTCATAAGGAT T (SEQ ID NO: 108) LFAAGCTACAGTGGCAAGAGAA (SEQ ID NO: 109) FB ATGCATCAGCTGTAGTGTTACT (SEQ ID NO: 110) MB-WT /5Cy3/GGAGCTT + T GT + CTGGTTT + TAAG + CTCC/3IAbRQSp/ (SEQ ID NO: 111) MB- /56-FAM/CGCAGTT + T DELGAAG + CTAAA + A + G A + CTGCG/3IABkFQ/ (SEQ ID NO: 112) AsleSEQ ID NOs: 46-51 E1 SEQ ID NOs: 70-75 N2 SEQ ID NOs: 52-57

LAMP primers and molecular beacons: LAMP primers were designed with theSGF deletion located between Bk and LB (FIGS. 37A and 37B) using LAMPPrimer Design Tool at NEB https://lamp.neb.com/#!/) and with enoughlength between Bk and LB to accommodate the location of molecularbeacons in this region. Molecular beacons targeting either wild-type orthe SGF deletion sequence were designed using principles according toSherrill-Mix et al., Genome Biol, 22(1), 169 (2021). As the deletion islocated in a relatively AT-rich region, the annealing temperatures forthese beacons are lower: the calculated Tm of the annealed beacon-targetfor wt MB and SGFdel MB is 63.9° C. and 62.5° C., and the stem is 55.1°C. and 54.7° C., respectively. These beacons were synthesized asAffinity Plus qPCR Probes by IDT with sequences shown in Table 9.

RT-LAMP reactions: RT-LAMP reactions were performed using WarmStart®LAMP Kit (DNA & RNA) (E1700) with standard primer concentrations (0.2 μMF3, 0.2 μM B3, 1.6 μM FIP, 1.6 μM BIP, 0.4 μM Loop F, 0.4 μM Loop B) inthe presence of 40 mM guanidine hydrochloride in 25 μl on 96-well platesat 65° C. in a Bio-Rad CFX96 instrument. LAMP amplification was measuredby including 1 uM SYTO™-9 fluorescent dye (ThermoFisher S34854), 0.5 μMSGFdel or 1.0 μM wt beacon and signal was acquired at 15 secondintervals. Synthetic SARS-CoV-2 RNAs were obtained from Twist Bioscience(Control 2 for WT MN908947.3, Control 14 for B.1.1.7, Control 16 forB.1.351 and Control 17 for P.1) and diluted in 10 ng/μl Jurkat total RNAbased on the copy number provided by the manufacturer.

Results

Positional Mutation Effects

To mimic the effect of a potential SARS-CoV-2 variant in an RT-LAMPassay, we focused on single point mutations at each primer base positionand the SGF deletion that is found in several variants of concern. Forthe single point mutation primers, each of the 527 variant primers fromthe 3 assays (As1e, N2, E1) was tested in RT-LAMP reactions with threedifferent SARS-CoV-2 RNA copy number concentrations: 100, 200, and10,000 copies in order to gain a sense of the mutation effect onreaction speed and sensitivity. Both lower concentrations allowed foramplification effects to be confidently determined outside of stochasticperformance when close to the limit of detection in the 100 copyreactions, particularly for the As1e primer set which displays slightlylower sensitivity in our testing. The reaction output speed was measuredrelative to the fully-complementary wild-type primer, and relativeeffect plotted against the position in the primer sequence (FIG.36A-36F). Overall, evaluating the 4,500 RT-LAMP assays with single pointmutations within any of the six primers, regardless of gene, resulted inminimal to no effect on the ability to amplify the target at any of thecopy numbers used.

The most common result across the primers was a 5%-10% reduction inamplification speed in the presence of a mismatch. The B3 primers showedremarkably little impact in any of the 69 variant primer sets for the 3amplicons, though the F3 primer did see some slowing when mismatcheswere present away from the 5′ end in the E1 and As1e sets (FIG. 36A,36B). These primers are the least critical to the reaction, but thedifference between the two may indicate differential tolerances tomismatches by the reaction initiation (B3 annealing to single-strandedRNA and extension by RTx reverse transcriptase) vs. the strand invasionand displacement via est 2.0 polymerase that occurs at the F3 primer.With the more critical FIP and BIP primers, the 3′ half of the primer(F2/B2) serves to invade and primer double-stranded DNA, with the 5′half annealing to displaced product strands to form the ‘loop’ dumbbellshapes for amplification. The results in FIG. 36C-D clearly indicatemore impact of mutations on the F2/B2 regions, with more variant oligoscausing amplification delays relative to fully base-paired controltoward the 3′ end of the FIP and BIP in all 3 gene assays. The extreme5′ end was more likely to display sensitivity to mismatch, likelyindicating an impact on extension from a mismatch in the looped-backLAMP hairpin structure, but sensitivity was not impacted in these tests.

As a summary of the effects Table 10 lists the number of positions fromeach primer that resulted in a detection cycle change of more than 10%from the WT primer. Though overall effects on amplification wereminimal, the greater impact of 3′ mutations is clear from the trends inFIG. 36A-F. And while a significant number of variant primers resultedin slowing of the reaction, in all 527 variant primers tested nomutation position prevented amplification in the RT-LAMP reaction withSARS-CoV-2 RNA even with lower RNA copy numbers.

TABLE 10 Effect of Single Point Mutations Primer No. Bases with >10%LAMP Time Change Total Bases F3 22 62 B3 0 69 FIP 27 131 BIP 21 128 LF18 68 LB 13 66Deletion Detection with Molecular Beacons

To investigate the effect of a deletion on an RT-LAMP assay, we designeda primer set targeting the SGF deletion region (Orf1a 3675-3677) withtwo molecular beacons targeting either the WT or the variant targetregion (FIG. 37A). We initially checked this primer set for specificityand sensitivity and found it was able to detect both WT and variants RNAwith similar sensitivity of approximately 50 copies and with no apparentnon-template amplification signal in 40 minute reactions (data notshown). We then evaluated detection using the molecular beacons with10-fold dilutions of WT or B.1.1.7 synthetic RNA and compared them withconventional intercalating fluorescence detection. Both molecularbeacons detected their intended targets as designed with robustspecificity and even at 10,000 copies of target RNA they only recognizedonly their intended amplification products (FIG. 37. B). This is incontrast to another strategy where the deletion was placed at the endsof the FIP and BIP [16] and detected with intercalating dye. With thisdesign, WT vs. B.1.1.7 discrimination was efficient at low (≤1,000copies RNA) inputs but the two variant sequences could only bedistinguished by their amplification speed at higher copy numbers.

We then assessed the sensitivity of the SGFdel beacon to detect variantRNA containing the SGF deletion. We performed 24 repeats of LAMPreactions each with 50 copies of synthetic RNAs from B.1.1.7, B.1.351,or P.1 and the reactions were detected by either the molecular beaconsor by dsDNA binding dye SYTO-9. The results indicated SGFdel MB was ableto detect all the variant RNAs with efficiency similar to that with dsDNA binding dye (Table 11). We further tested combining both molecularbeacons so that the identity of the input viral RNA could be determinedin the same LAMP reaction. We tested 24 repeat reactions in the presenceof both SGFwt or SGFdel molecular beacons and compared to the detectionwith single beacons. We found that target RNAs were reliably detectedwith the same level of sensitivity when both beacons were present and nocross interaction between the two beacons was observed (Table 12).

TABLE 11 Specific Detection of Variant RNA with LAMP and MolecularBeacons RNA WT B.1.1.7 B.1.351 P1 SYTO-9 21 21 23 24 Beacon 18 17 24 23positives from 24 repeats, 50 copies/reaction

TABLE 12 Dual-beacon RT-LAMP for Variant RNA Detection Single BeaconDuplex Cy3 (WT) FAM (Del) Cy3 (WT) FAM (Del) RNA WT 20 — 20 0 P1 — 18 022 positives from 24 repeats, 50 copies/reaction

Here we established the first comprehensive screen of LAMP primertolerance to mutation in target nucleic acids with (i) a known sequenceor (ii) a known sequence with a plurality of undefined mutationstherein.

We investigated a single base mutation at every position of every primerin three prominent SARS-CoV-2 RT-LAMP assays. Remarkably, we find verylittle impact of the single base changes, with only marginal effect onspeed in most positions. The robustness of RT-LAMP to sequence variationis a significant benefit to its adoption, with reduced worry aboutdeleterious effects from the commonly emerging single-base changes thatcould occur with some frequency in the regions targeted by the LAMPprimers. Additionally, many RT-LAMP assays combine primer sets for addedspeed and sensitivity [4], adding an additional layer of protectionagainst possible sequence variation.

The converse of this assay robustness however is an inherent difficultyin identifying variants during the amplification reaction. Whilesequencing offers greater confidence and detail for variant calling theability to utilize the diagnostic amplification for prospective variantidentification as with the TaqPath S-gene dropout is a valuable featureof a potential diagnostic method. We observed difficulty targeting evena large 9-base deletions by typical LAMP primer design alone, but byutilizing a molecular beacon approach as first described by [19] we wereable to accurately amplify and identify RNA from the three SARS-CoV-2variant sequences containing the SGF deletion. By combining the beaconsfor the wild-type and deletion sequence, we could call wild-type orvariant based on the detected sequence, indicating the potential abilityfor variant calling in the RT-LAMP assay by multiplexed beacon design.

Taken together these data position RT-LAMP as an attractive diagnosticmethod with a high level of tolerance to common sequence mutations.Recent FDA guidance described a need for understanding this tolerancefor any molecular diagnostic test and use of RT-LAMP provides increasedconfidence in a diagnostic test that detects evolving pathogens withconsistent performance. In situations where variant identification isdesired, use of molecular beacons provides a sensitive addition tospecific sequence detection with fluorescence detection.

What is claimed is:
 1. A kit for use in diagnostic detection of a targetnucleic acid and variants thereof having undefined mutations, obtainedfrom a cell or virus in a biological sample, the kit comprising: (a) alyophilized mixture of a strand displacing polymerase and an indicatorreagent and optionally a lyophilized reverse transcriptase, wherein: (i)the indicator reagent is characterized by its ability to change color orprovide fluorescence in a nucleic acid amplification reaction; and (ii)the strand displacing polymerase when rehydrated is capable ofamplifying DNA at a temperature in the range of 50° C.-68° C.; (b) auniversal primer set suitable for loop mediated amplification (LAMP) ofthe target nucleic acids and variants thereof containing undefinedmutations within one or more of the primer binding sites; wherein any ofthe reagents in the kit may be combined in a mixture in a singlecontainer or provided in separate containers.
 2. The kit according toclaim 1, wherein the universal primer set suitable for LAMP is capableof hybridizing to the target DNA in the presence of a plurality ofundefined mutations to provide a positive result for the target DNA in apredetermined assay time period otherwise determined for a positivesample of a target nucleic acid having a known sequence.
 3. The kitaccording to claim 2, wherein the universal primer set suitable for loopmediated amplification (LAMP) are similarly diagnostic for the targetnucleic acids and variants thereof where the any deletions and additionsin the BIF and FIP primer binding sites of the variants do not exceed6-9 nucleotides.
 4. The kit according to claim 1, wherein the target DNAis the reverse transcription product of an RNA virus.
 5. The kitaccording to claim 4, wherein the RNA virus is a coronavirus.
 6. The kitaccording to claim 1, wherein the indicator reagent is a molecularbeacon or a metallochromic dye.
 7. The kit according to claim 1, furthercomprising (c) a lysis reagent in a container for receiving thebiological sample, wherein the lysis reagents comprise a reducing agentand a metal chelator.
 8. The kit according to claim 7, wherein thereducing agent is Tris (2-carboxyethyl) phosphine hydrochloride (TCEP).9. The kit according to any of claim 1, wherein the lysis reagentscomprise at least one of a salt of C—(NH₂)₂NH⁺ and a poloxamer.
 10. Thekit according to claim 1, wherein one or more of components in (a)-(b)are immobilized on a substrate.
 11. The kit according to claim 1,comprising the reverse transcriptase, wherein the reverse transcriptaseis a virus encoded reverse transcriptase, or a bacteria encoded intronII reverse transcriptase.
 12. A method for detecting a target nucleicacid or unknown variant thereof in a biological sample by Loop-MediatedIsothermal Amplification (LAMP), comprising: (a) combining thebiological sample with a lysis reagent to form a lysis mix; (b)incubating the lysis mix at a temperature of at least 60° C. for aperiod of time in the range of 2 minutes to 45 minutes; (c) combining ina reaction mix, an aliquot of the heat treated lysis mix of step (b)with amplification reagents comprising a strand displacing polymerase, areversible inhibitor of the polymerase, nucleoside triphosphates, and atleast one set of LAMP primers that is capable of hybridizing to thetarget nucleic acid and to undefined variants of the target nucleicacid; and (d) incubating the reaction mix for a reaction positive periodof time under amplification conditions for LAMP to detect the presenceof the target nucleic acid or undefined variants thereof in the sample.13. The method of claim 12, wherein the lysis reagent comprises areducing agent and a metal chelating reagent.
 14. The method of claim12, wherein (b) further comprises incubating the lysis mix at 95° C. for5 minutes.
 15. The method of claim 12, wherein the amplificationreagents include a reverse transcriptase and a reversible inhibitor ofthe reverse transcriptase.
 16. The method according to claim 12, whereinany of the reagents in the lysis mix and any of the amplificationreagents may be lyophilized prior to combining with the biologicalsample.
 17. The method according to claim 12, wherein any of thereagents in the lysis mix and any of the amplification reagents may beimmobilized on a matrix.
 18. The method according to claim 12, whereinthe biological sample is saliva.
 19. The method according to claim 12,wherein the target nucleic acid is an RNA virus.
 20. The methodaccording to claim 19, wherein the RNA virus is a coronavirus and theamplification reagents include multiple sets of universal primers. 21.The method according to claim 12, further comprising step (e) sequencingthe detected target nucleic acid or variants thereof to determine thepresence of a novel mutations.